Streptococcus bacteriophage lysins for detection and treatment of gram positive bacteria

ABSTRACT

The present invention provides methods, compositions and articles of manufacture useful for the prophylactic and therapeutic amelioration and treatment of gram-positive bacteria, including Streptococcus and Staphylococcus, and related conditions. The invention provides compositions and methods incorporating and utilizing Streptococcus suis derived bacteriophage lysins, particularly PlySs2 and/or PlySs1 lytic enzymes and variants thereof, including truncations thereof. Methods for treatment of humans are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of co-pending application Ser.No. 15/492,128, filed Apr. 20, 2017, now U.S. Pat. No. 10,544,407,issued Jan. 28, 2020, which is a Continuation of co-pending applicationSer. No. 14/685,696, filed Apr. 14, 2015, now U.S. Pat. No. 9,914,915,issued Mar. 13, 2018, which is a Continuation of National Stageapplication Ser. No. 14/112,963, filed Nov. 19, 2013, now U.S. Pat. No.9,034,322, issued May 19, 2015, which claims priority from PCTApplication No. PCT/US2012/034456 filed Apr. 20, 2012, which in turn,claims priority from U.S. Provisional Application Ser. No. 61/477,836filed Apr. 21, 2011. Applicants claim the benefits of 35 U.S.C. § 120 asto the National Stage Application and the PCT application and priorityunder 35 U.S.C. § 119 as to the said U.S. Provisional application, andthe entire disclosures of all applications are incorporated herein byreference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberAI011822 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE

The specification is amended to direct entry of the Sequence Listing inthe ASCII text file having i) the name2729-11PCTUSCON2DIV_Sequence_Listing; ii) a date of creation of Jan. 30,2020; and iii) a size of 19.9 kilobytes into the application. Thematerial in that ASCII text file having i) the name2729-11PCTUSCON2DIV_Sequence_Listing; ii) a date of creation of Jan. 30,2020; and iii) a size of 19.9 kilobytes is incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates generally to methods, compositions andarticles of manufacture useful for the prophylactic and therapeuticamelioration and treatment of gram-positive bacteria, includingStreptococcus and Staphylococcus bacterial strains, including pathogenicand antibiotic-resistant bacteria, and related conditions. The inventionrelates to compositions and articles of manufacture incorporatingisolated Streptococcus suis bacteriophage lysins including PlySs2 and/orPlySs1 lytic enzymes and variants thereof, including truncationsthereof, and to methods utilizing the lysin polypeptides andcompositions.

BACKGROUND OF THE INVENTION

A major problem in medicine has been the development of drug resistantbacteria as more antibiotics are used for a wide variety of illnessesand other conditions. Hospital infections are the 8′ leading cause ofdeath in the United States, due in large part to drug-resistant andnewly-emerging pathogens. For example, there are over 500,000 cases ofStaphylococcus aureus annually in the U.S. and over 65% of strains aremultidrug resistant (MRSA). The use of more antibiotics and the numberof bacteria showing resistance has prompted longer treatment times.Furthermore, broad, non-specific antibiotics, some of which havedetrimental effects on the patient, are now being used more frequently.A related problem with this increased use is that many antibiotics donot penetrate mucus linings easily. Additionally, the number of peopleallergic to antibiotics appears to be increasing. Accordingly, there isa commercial need for new antibacterial approaches, especially thosethat operate via new modalities or provide new means to kill pathogenicbacteria.

Gram-positive bacteria are surrounded by a cell wall containingpolypeptides and polysaccharide. The gram-positive cell wall appears asa broad, dense wall that is 20-80 nm thick and consists of numerousinterconnecting layers of peptidoglycan. Between 60% and 90% of thegram-positive cell wall is peptidoglycan, providing cell shape, a rigidstructure, and resistance to osmotic shock. The cell wall does notexclude the Gram stain crystal violet, allowing cells to be stainedpurple, and therefore “Gram-positive.” Gram-positive bacteria includebut are not limited to the genera Actinomyces, Bacillus, Listeria,Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium,Corynebacterium, and Clostridium. Medically relevant species includeStreptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus,and Enterococcus faecalis. Bacillus species, which are spore-forming,cause anthrax and gastroenteritis. Spore-forming Clostridium species areresponsible for botulism, tetanus, gas gangrene and pseudomembranouscolitis. Corynebacterium species cause diphtheria, and Listeria speciescause meningitis.

Antibacterials that inhibit cell wall synthesis, such as penicillins andcephalosporins, interfere with the linking of the interpeptides ofpeptidoglycan and weaken the cell wall of both gram positive and gramnegative bacteria. Because the peptidoglycans of gram-positive bacteriaare exposed, gram-positive bacteria are more susceptible to theseantibiotics. Advantageously, eukaryotic cells lack cell walls and arenot susceptible to these drugs or other cell wall agents.

Attempts have been made to treat bacterial diseases through the use ofbacteriophages. However, the direct introduction of bacteriophages intoan animal to prevent or fight diseases has certain drawbacks.Specifically, both the bacteria and the phage have to be in the correctand synchronized growth cycles for the phage to attach. Additionally,there must be the right number of phages to attach to the bacteria; ifthere are too many or too few phages, there will be either no attachmentor no production of the lysing enzyme. The phage must also be activeenough. The phages are also inhibited by many things including bacterialdebris from the organism it is going to attack. Further complicating thedirect use of a bacteriophage to treat bacterial infections is thepossibility of immunological reactions, rendering the phagenon-functional.

Novel antimicrobial therapy approaches include enzyme-based antibiotics(“enzybiotics”) such as bacteriophage lysins. Phages use these lysins todigest the cell wall of their bacterial hosts, releasing viral progenythrough hypotonic lysis. A similar outcome results when purified,recombinant lysins are added externally to Gram-positive bacteria. Thehigh lethal activity of lysins against Gram-positive pathogens makesthem attractive candidates for development as therapeutics.Bacteriophage lysins were initially proposed for eradicating thenasopharyngeal carriage of pathogenic streptococci (Loeffler, J. M. etal (2001) Science 294: 2170-2172; Nelson, D. et al (2001) Proc Natl AcadSci USA 98:4107-4112). Lysins are part of the lytic mechanism used bydouble stranded DNA (dsDNA) phage to coordinate host lysis withcompletion of viral assembly (Wang, I. N. et al (2000) Annu RevMicrobiol 54:799-825). Phage encode both holins that open a pore in thebacterial membrane, and peptidoglycan hydrolases called lysins thatbreak bonds in the bacterial wall [6]. Late in infection, lysintranslocates into the cell wall matrix where it rapidly hydrolyzescovalent bonds essential for peptidoglycan integrity, causing bacteriallysis and concomitant progeny phage release.

Lysin family members exhibit a modular design in which a catalyticdomain is fused to a specificity or binding domain (Lopez, R. et al(1997) Microb Drug Resist 3:199-211). Lysins can be cloned from viralprophage sequences within bacterial genomes and used for treatment(Beres, S. B. et al (2007) PLoS ONE 2(8):1-14). When added externally,lysins are able to access the bonds of a Gram-positive cell wall(FIG. 1) (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400).Lysins have been shown to demonstrate a high lethal activity againstnumerous Gram-positive pathogens (especially the bacterium from whichthey were cloned), raising the possibility of their development astherapeutics (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400;Nelson, D. L. et al (2001) Proc Natl Acad Sci USA 98:4107-4112).

Bacteriophage lytic enzymes have been established as useful in theassessment and specific treatment of various types of infection insubjects through various routes of administration. For example, U.S.Pat. No. 5,604,109 (Fischetti et al.) relates to the rapid detection ofGroup A streptococci in clinical specimens, through the enzymaticdigestion by a semi-purified Group C streptococcal phage associatedlysin enzyme. This enzyme work became the basis of additional research,leading to methods of treating diseases. Fischetti and Loomis patents(U.S. Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use ofa lysin enzyme produced by group C streptococcal bacteria infected witha C1 bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and Loomis)discloses a composition for dermatological infections by the use of alytic enzyme in a carrier suitable for topical application to dermaltissues. U.S. Pat. No. 6,254,866 (Fischetti and Loomis) discloses amethod for treatment of bacterial infections of the digestive tractwhich comprises administering a lytic enzyme specific for the infectingbacteria. The carrier for delivering at least one lytic enzyme to thedigestive tract is selected from the group consisting of suppositoryenemas, syrups, or enteric coated pills. U.S. Pat. No. 6,264,945(Fischetti and Loomis) discloses a method and composition for thetreatment of bacterial infections by the parenteral introduction(intramuscularly, subcutaneously, or intravenously) of at least onelytic enzyme produced by a bacteria infected with a bacteriophagespecific for that bacteria and an appropriate carrier for delivering thelytic enzyme into a patient.

Phage associated lytic enzymes have been identified and cloned fromvarious bacteriophages, each shown to be effective in killing specificbacterial strains. U.S. Pat. Nos. 7,402,309, 7,638,600 and published PCTApplication WO2008/018854 provides distinct phage-associated lyticenzymes useful as antibacterial agents for treatment or reduction ofBacillus anthracis infections. U.S. Pat. No. 7,569,223 describes lyticenzymes for Streptococcus pneumoniae. Lysin useful for Enterococcus (E.faecalis and E. faecium, including vancomycin resistant strains) aredescribed in U.S. Pat. No. 7,582,291. US 2008/0221035 describes mutantPly GBS lysins highly effective in killing Group B streptococci. Achimeric lysin denoted ClyS, with activity against Staphylococcibacteria, including Staphylococcus aureus, is detailed in WO2010/002959.

Streptococcus suis is a Gram-positive pathogen that infects pigsworldwide. Reports of zoonotic transmission from pigs to humans areincreasing (Sriskandan S. et al (2006) PLoS Medicine 3(5):585-567). S.suis may develop a consistent presence in human populations in years tocome. Humans and pigs have been treated with penicillin or gentamicin,but S. suis isolates resistant to these antibiotics exist (Cantin, M. etal (1992) J Vet Diagnostic Investig 4:170-174).

It is evident from the deficiencies and problems associated with currenttraditional antibacterial agents that there still exists a need in theart for additional specific bacterial agents and also for broaderspectrum agents, particularly without high risks of acquired resistance.It is notable that to date, no lysin has been shown to demonstrate broadlytic activity against multiple distinct species of pathogenic andclinically relevant bacteria.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention provides a lysin havingbroad killing activity against multiple bacteria, particularlygram-positive bacteria, including Staphylococcus, Streptococcus,Enterococcus and Listeria bacterial strains. The invention provides abacteriophage lysin capable of killing bacteria from distinct orders. Inan apect, a lysin polypeptide is provided capable of killing one or morebacteria from distinct orders of Bacilli, particularly order Bacilallesand order Lactobacillales. The present invention provides lysinpolypeptide capable of and demonstrated to kill bacteria from twodistinct orders, particularly Bacillales and Lactobacillales, in vitroand in vivo. Lysin of the present invention is capable of killingBacillales and Lactobacillales bacteria in mixed culture and in mixedinfections in vivo. The invention thus contemplates treatment,decolonization, and/or decontamination of bacteria, cultures orinfections or in instances wherein more than one gram positive bacteria,particularly one or more of Staphylococcus, Streptococcus, Enterococcusand Listeria bacteria, is suspected or present. In particular, theinvention contemplates treatment, decolonization, and/or decontaminationof bacteria, cultures or infections or in instances wherein more thanone type of Bacilalles bacteria, more than one type of Lactobacillalesbacteria, or at least one type of Bacillales and one type ofLactobacillales bacteria is suspected, present, or may be present.

In accordance with the present invention, bacteriophage lysins areprovided which are derived from Streptococcus suis bacteria. Twoexemplary distinct and unique lysins have been isolated andcharacterized, particularly PlySs1, including an active truncationthereof, and PlySs2. The lysin polypeptides of the present invention areunique in demonstrating broad killing activity against multiplebacteria, particularly gram-positive bacteria, including Staphylococcus,Streptococcus, Enterococcus and Listeria bacterial strains. In one suchaspect, the PlySs2 lysin is capable of killing Staphylococcus aureusstrains and bacteria in animal models, as demonstrated herein in mice.PlySs2 is effective against antibiotic-resistant Staphylococcus aureussuch as methicillin-resistant Staphylococcus aureus (MRSA), vancomycinintermediate-sensitivity Staphylococcus aureus (VISA), and vancomycinresistant Staphylococcus aureus (VRSA). In a further such aspect, thePlySs1 lysin is capable of reducing growth of Staphylococcus aureusstrains and bacteria, including antibiotic-resistant Staphylococcusaureus such as methicillin-resistant Staphylococcus aureus (MRSA),vancomycin intermediate-sensitivity Staphylococcus aureus (VISA), orvancomycin resistant Staphylococcus aureus (VRSA). The inventionincludes compositions and articles of manufacture comprising the lysinpolypeptides and methods of prevention and treatment of bacterialgrowth, colonization and infections.

In an aspect of the invention, a method is provided of killinggram-positive bacteria comprising the step of contacting the bacteriawith a composition comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, the isolated lysin polypeptidecomprising the PlySs2 lysin polypeptide or variants thereof effective tokill gram-positive bacteria.

Thus, a method is provided of killing gram-positive bacteria comprisingthe step of contacting the bacteria with a composition comprising anamount of an isolated lysin polypeptide effective to kill thegram-positive bacteria, the isolated lysin polypeptide comprising theamino acid sequence of SEQ ID NO:3 or variants thereof having at least80% identity, 85% identity, 90% identity, 95% identity or 99% identityto the polypeptide of SEQ ID NO:3 and effective to kill thegram-positive bacteria.

In an additional aspect of the above method, the composition furthercomprises an effective amount of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:1, the isolated lysinpolypeptide comprising the amino acid sequence of SEQ ID NO:2, orvariants thereof having at least 80% identity to the polypeptide of SEQID NO:1 or of SEQ ID NO:2 and effective to kill the gram-positivebacteria.

The invention provides a method of killing gram-positive bacteriacomprising the step of contacting the bacteria with a compositioncomprising an amount of an isolated lysin polypeptide effective to killgram-positive bacteria, the isolated lysin polypeptide comprising thePlySs1 lysin polypeptide or truncations or variants thereof effective tokill gram-positive bacteria. In an aspect of this method, thecomposition comprises an effective amount of the isolated lysinpolypeptide comprising the amino acid sequence of SEQ ID NO:1, theisolated truncated lysin polypeptide comprising the amino acid sequenceof SEQ ID NO:2, or variants thereof having at least 80% identity, 85%identity, 90% identity, 95% identity or 99% identity to the polypeptideof SEQ ID NO:1 or of SEQ ID NO:2 and effective to kill the gram-positivebacteria.

In an aspect of the above methods of killing gram positive bacteria, themethods are performed in vitro or ex vivo so as to sterilize ordecontaminate a solution, material or device, particularly intended foruse by or in a human.

The invention provides a method for reducing a population ofgram-positive bacteria comprising the step of contacting the bacteriawith a composition comprising an amount of an isolated polypeptideeffective to kill at least a portion of the gram-positive bacteria, theisolated polypeptide comprising the amino acid sequence of SEQ ID NO:3or variants thereof having at least 80% identity to the polypeptide ofSEQ ID NO:3 and effective to kill the gram-positive bacteria. In anembodiment of this method, the composition further comprises aneffective amount of the isolated lysin polypeptide comprising the aminoacid sequence of SEQ ID NO:1, the isolated lysin polypeptide comprisingthe amino acid sequence of SEQ ID NO:2, or variants thereof having atleast 80% identity to the polypeptide of SEQ ID NO:1 or of SEQ ID NO:2and effective to kill the gram-positive bacteria.

The invention further provides a method for reducing a population ofgram-positive bacteria comprising the step of contacting the bacteriawith a composition comprising an amount of an isolated polypeptideeffective to kill at least a portion of the gram-positive bacteria, theisolated polypeptide comprising the PlySs1 lysin polypeptide ortruncations or variants thereof effective to kill gram-positivebacteria. In an aspect of this method, the composition comprises aneffective amount of the isolated lysin polypeptide comprising the aminoacid sequence of SEQ ID NO: 1, the isolated lysin polypeptide comprisingthe amino acid sequence of SEQ ID NO:2, or variants thereof having atleast 80% identity, 85% identity, 90% identity or 95% identity to thepolypeptide of SEQ ID NO:1 or of SEQ ID NO:2 and effective to kill thegram-positive bacteria.

In an aspect of the above methods for reducing a population of grampositive bacteria, the methods are performed in vitro or ex vivo so asto sterilize or decontaminate a solution, material or device,particularly intended for use by or in a human.

The present invention further provides a method for treating anantibiotic-resistant Staphylococcus aureus infection in a humancomprising the step of administering to a human having anantibiotic-resistant Staphylococcus aureus infection, an effectiveamount of a composition comprising an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:3 or variants thereof having atleast 80% identity, 85% identity, 90% identity or 95% identity to thepolypeptide of SEQ ID NO:3 and effective to kill Staphylococcus aureus,whereby the number of Staphylococcus aureus in the human is reduced andthe infection is controlled.

In an aspect of this method, the composition may alternatively or mayfurther comprise an effective amount of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:1, the isolated lysinpolypeptide comprising the amino acid sequence of SEQ ID NO:2, orvariants thereof having at least 80% identity, 85% identity, 90%identity or 95% identity to the polypeptide of SEQ ID NO:1 or of SEQ IDNO:2 and effective to kill Staphylococcus aureus.

A method of the invention also includes a method for treatinggram-positive bacterial infection caused by one or more ofStaphylococcus, Streptococcus, Enterococcus or Listeria bacteria in ahuman comprising the step of administering to a subject having abacterial infection, an effective amount of a composition comprising anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:3or variants thereof having at least 80% identity, 85% identity, 90%identity or 95% identity to the polypeptide of SEQ ID NO:3 and effectiveto kill the gram-positive bacteria, whereby the number of gram-positivebacteria in the human is reduced and the infection is controlled.

The composition of use in the above method may alternatively or mayfurther comprise an effective amount of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:1, the isolated lysinpolypeptide comprising the amino acid sequence of SEQ ID NO:2, orvariants thereof having at least 80% identity, 85% identity, 90%identity or 95% identity to the polypeptide of SEQ ID NO:1 or of SEQ IDNO:2 and effective to kill the gram-positive bacteria.

The invention additionally includes a method for treating a humansubject exposed to or at risk for exposure to a pathogenic gram-positivebacteria comprising the step of administering to the subject acomposition comprising an amount of an isolated lysin polypeptideeffective to kill the gram-positive bacteria, the isolated lysinpolypeptide comprising the amino acid sequence of SEQ ID NO:3 orvariants thereof having at least 80% identity, 85% identity, 90%identity or 95% identity to the polypeptide of SEQ ID NO:3 and effectiveto kill the gram-positive bacteria. In a particular aspect of thismethod, wherein the subject is exposed to or at risk of one of or one ormore of Staphylococcus (such as Staphylococcus aureus), Streptococcus(such as Streptococcus pyogenes), Listeria (such as L. monocytogenes),or Enterococcus (such as E. faecalis) bacteria. The subject may be ahuman. The subject may be a human adult, child, infant or fetus.

Variants of a lysin polypeptide of use in the compositions and methodsof the invention may be substantially identical to one or more of thelysin polypeptide(s) exemplified herein, including to SEQ ID NO: 1, 2 or3. Variants of a lysin polypeptide of use in the compositions andmethods of the invention may have at least 75% identity, at least 80%identity, at least 90% identity, at least 95% identity in amino acidsequence as compared to the lysin polypeptide(s) exemplified herein,including to SEQ ID NO: 1, 2 or 3.

In any such above method or methods, the susceptible, killed or treatedbacteria may be selected from Staphylococcus aureus, Listeriamonocytogenes, Staphylococcus simulans, Streptococcus suis,Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo,Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS),Streptococcus sanguinis, Streptococcus gordonii, Streptococcusdysgalactiae, Group G Streptococcus, Group E Streptococcus, Enterococcusfaecalis and Streptococcus pneumonia.

In accordance with any of the methods of the invention, the susceptiblebacteria or bacteria being treated or decolonized may be an antibioticresistant bacteria. The bacteria may be methicillin-resistantStaphylococcus aureus (MRSA), vancomycin intermediate-sensitivityStaphylococcus aureus (VISA), or vancomycin resistant Staphylococcusaureus (VRSA). The susceptible bacteria may be a clinically relevant orpathogenic bacteria, particularly for humans. In an aspect of themethod(s), the lysin polypeptide(s) is effective to kill Staphylococcus,Streptococcus, Enterococcus and Listeria bacterial strains.

In accordance with any of the methods of the invention, the compositionthereof may further comprise a carrier, including a pharmaceuticallyacceptable carrier, additive or diluent. In accordance with any of themethods of the invention, the composition thereof may further comprise asuitable vehicle for delivery of the polypeptide to a site of infection.In accordance with any of the methods of the invention, the compositionthereof may further comprise one or more antibiotic.

The invention provides compositions, including thereapeutic andpharmaceutical compositions comprising one or more lysin polypeptide ofthe invention.

The invention thus provides a pharmaceutical composition for killinggram-positive bacteria comprising the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:3 andeffective to kill the gram-positive bacteria.

In an embodiment, the pharmaceutical composition may alternatively ormay further comprise an effective amount of the isolated lysinpolypeptide comprising the amino acid sequence of SEQ ID NO: 1, theisolated lysin polypeptide comprising the amino acid sequence of SEQ IDNO:2, or variants thereof having at least 80% identity to thepolypeptide of SEQ ID NO:1 or of SEQ ID NO:2 and effective to kill thegram-positive bacteria.

In an aspect of the invention, a pharmaceutical composition is providedfor killing gram-positive bacteria comprising at least two isolatedlysin polypeptides wherein the first isolated polypeptide comprises theamino acid sequence of SEQ ID NO:3 or variants thereof having at least80% identity to the polypeptide of SEQ ID NO:3 and effective to kill thegram-positive bacteria, and the second isolated polypeptide comprisesthe amino acid sequence of SEQ ID NO: 1, the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:2, or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:1 or of SEQID NO:2 and effective to kill the gram-positive bacteria.

In a further aspect thereof, a composition, including a therapeutic orpharmaceutical composition, may comprise a truncated lysin having theamino acid sequence of SEQ ID NO: 2 with a modification whereby thetruncated lysin comprises only one catalytic domain selected from thegroup consisting of an endopeptidase domain and a glucosaminidasedomain. In an additional aspect of the composition, the truncated lysindoes not include the glucosaminidase domain of SEQ ID NO:1. Thetruncated lysin may particularly have the amino acid sequence of SEQ IDNO:2, or variants thereof having at least 80% identity to thepolypeptide of SEQ ID NO:2 and effective to kill gram-positive bacteria.

The invention includes an article of manufacture comprising a vesselcontaining a composition comprising an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:3, or variants thereof having atleast 80% identity, 85% identity, 90% identity or 95% identity to thepolypeptide of SEQ ID NO:3 and effective to kill gram-positive bacteria,and instructions for use of the composition in treatment of a patientexposed to or exhibiting symptoms consistent with exposure toStaphylococcus, Streptococcus or Listeria bacteria, where theinstructions for use of the composition indicate a method for using thecomposition, the method comprising the steps of:

-   -   a) identifying the patient suspected of having been exposed to        Staphylococcus, Streptococcus or Listeria bacteria; and    -   b) administering an effective amount of the composition to the        patient.

In one aspect of the article of the invention, the isolated polypeptideof the composition has the amino acid sequence of SEQ ID NO:3. In anadditional aspect of the article of the invention, the compositionalternatively or further comprises an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQID NO:2, or variants thereof having at least 80% identity, 85% identity,90% identity or 95% identity to the polypeptide of SEQ ID NO:1 or of SEQID NO:2 and effective to kill gram-positive bacteria.

The compositions of the invention may particularly demonstrate or havekilling activity against one or more bacteria strains, particularlyselected from the group consisting of Staphylococcus aureus, Listeriamonocytogenes, Staphylococcus simulans, Streptococcus suis,Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo,Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS),Streptococcus sanguinis, Streptococcus gordonii, Streptococcusdysgalactiae, Group G Streptococcus, Group E Streptococcus, Enterococcusfaecalis and Streptococcus pneumonia.

The present invention also provides nucleic acids encoding the lysinpolypeptides of the invention. Thus, nucleic acids are provided encodingS. suis lysins PlySs1, truncated or whole lysin, and PlySs2. Exemplarynucleic acid sequences are provided in FIG. 3 and in FIG. 4. Nucleicacids capable of encoding a polypeptide of SEQ ID NO:1, SEQ ID NO:2 orSEQ ID NO:3, including variants thereof are provided herein.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes an S. suislysin or lysin polypeptide; preferably a nucleic acid molecule, inparticular a recombinant DNA molecule or cloned gene, encoding thePlySs1 lysin polypeptide, truncated or whole lysin, and/or the PlySs2lysin polypeptide, has a nucleotide sequence or is complementary to aDNA sequence shown in FIG. 3 and in FIG. 4.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule, cloned gene, or nucleic acid sequence encodinga lysin polypeptide hereof may be operatively linked to an expressioncontrol sequence which may be introduced into an appropriate host. Theinvention accordingly extends to unicellular hosts, including bacterialhosts, transformed with the nucleic acid sequence, cloned gene orrecombinant DNA molecule comprising a DNA sequence encoding the presentlysin polypeptide(s), and more particularly, the complete DNA sequencedetermined from the sequences set forth above and in FIG. 3 and FIG. 4.

The present invention naturally contemplates several means forpreparation of the lysin polypeptide(s), including as illustrated hereinknown recombinant techniques, and the invention is accordingly intendedto cover such synthetic preparations within its scope. The isolation ofthe DNA and amino acid sequences disclosed herein facilitates thereproduction of the lysin polypeptide(s) by such recombinant techniques,and accordingly, the invention extends to expression vectors preparedfrom the disclosed DNA sequences for expression in host systems byrecombinant DNA techniques, and to the resulting transformed hosts.

According to other preferred features of certain embodiments of thepresent invention, a recombinant expression system is provided toproduce biologically active lysin polypeptide(s). A process forpreparation of the polypeptides, particularly one or more lysinpolypeptide of the invention, is provided comprising culturing a hostcell containing an expression vector encoding one or more lysinpolypeptide(s) of the invention or capable of expressing a lysinpolypeptide(s) of the invention, and recovering the polypeptide(s).

The diagnostic utility of the present invention extends to the use ofthe present lysin polypeptides in assays to screen for the presence ofgram-positive bacteria, to screen for the presence of susceptiblegram-positive bacteria, or to determine the susceptibility of bacteriato killing or lysing by a one or more lysin polypeptide(s) of theinvention.

The present invention extends to the development of antibodies againstthe lysin polypeptide(s), or alternatively against the cleavage targetof the lysin polypeptide, including naturally raised and recombinantlyprepared antibodies. Such antibodies could include both polyclonal andmonoclonal antibodies prepared by known genetic techniques, as well asbi-specific (chimeric) antibodies, and antibodies including otherfunctionalities suiting them for additional diagnostic use conjunctivewith their capability of modulating lysin activity.

Lysin polypeptides which are modified and are chimeric or fusionproteins, or which are labeled, are contemplated and provided herein. Ina chimeric or fusion protein, the lysin polypeptide(s) of the inventionmay be covalently attached to an entity which may provide additionalfunction or enhance the use or application of the lysin polypeptide(s),including for instance a tag, label, targeting moiety or ligand, a cellbinding or cell recognizing motif or agent, an antibacterial agent, anantibody, an antibiotic. Exemplary labels include a radioactive label,such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe,⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. The label may be an enzyme, and detection ofthe labeled lysin polypeptide may be accomplished by any of thepresently utilized or accepted colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the lytic cycle versus lysin treatment. Lysinsrecombinantly expressed and purified are able to lyse bacteria just aswell as phage expressing lysins from within their host.

FIG. 2 depicts the PlySs2 domains. The catalytic domain corresponds toresidues 8-146. There is a 16-residue linker. The binding domaincorresponds to residues 162-228.

FIGS. 3A, 3B and 3C provides the (A) nucleotide and (B) amino acidsequence of the lysin PlySs1 as well as (C) a protein domain analysis.The amino acid sequence of the full length PlySs1 (SEQ ID NO:1) andtruncated PlySs1 (SEQ ID NO:2) are provided. The endopeptidase domain(SEQ ID NO:6), dual CPL-7 domain (SEQ ID NO:7) and glucosaminidasedomain (SEQ ID NO:8) denoted.

FIGS. 4A, 4B and 4C provides the (A) nucleotide and (B) amino acidsequence of the lysine PlySs2 as well as a (C) protein domain analysis.The amino acid sequence of PlySs2 corresponds to SEQ ID NO:3. The CHAPdomain and the SH-3 domain of the PlySs2 lysin are shaded, with the CHAPdomain starting with LNN . . . and ending with . . . YIT (SEQ ID NO:4)and the SH-3 domain starting with RSY . . . and ending with . . . VAT(SEQ ID NO:5).

FIG. 5 depicts the pBAD24 vector. The sequence begins with the pBADarabinose-inducible promoter for the T7 polymerase and ends with PlySs2.Ampicillin serves as a selective marker to ensure retention of theplasmid as cells grow.

FIG. 6 shows PlySs2 purification. All samples were run on 4-12% Bis-Trisgels at 200 V for ˜40 mins and stained with Coomassie. A. The DEAEcolumn flow through containing PlySs2 at ˜26 kDa. B. Six representativefractions of PlySs2 purified from a 10 L prep. C. A single band at ˜26kDa indicating the purity of PlySs2 after all fractions were pooledtogether.

FIG. 7A-7D depicts various aspects of PlySs2 characterization. A. Totest the optimal pH for PlySs2 activity, 50 μL of variousphosphate/citrate buffers pH levels were mixed with 195 μL S. suisstrain 7997 cells and 5 μL of lysin. PlySs2 had the strongest activityat pH 8.0. PlySs2 was shown to have acute activity up to pH 9.7. B. 195μL of cells, 5 μL lysin were added to 50 μL of various NaClconcentrations to determine the optimal salt concentration for PlySs2.C. To determine the temperature stability of PlySs2, it was incubatedfor 30 minutes at various temperatures, cooled and then added to 245 μLcells suspended in 15 mM Na₃PO₄, pH 8.0. D. PlySs2 was added to cellssuspended in 15 mM Na₃PO₄, pH 8.0 along with various concentrations ofethylenediaminetetraacetate (EDTA) to determine if it requires acofactor. In controls, dd H₂O replaced PlySs2 for all tests.

FIG. 8 depicts optimal pH of PlySs2 determined against S. suis strain7997 in Bis-tris propane (BTP) buffer up to a higher pH level.

FIG. 9. The stability of purified PlySS2 was determined by evaluatingkilling effectiveness against strain 7997 after storage at 37° C. for upto 48 hours in buffer.

FIG. 10. Killing effectiveness, assessed by OD₆₀₀ growth of strain 7997upon treatment with PlySs2 lysin after lysin storage at −80° C. for upto 7 months in buffer.

FIGS. 11A and 11B depicts ΔPlySs1 pH dependence. (A) Cells of hoststrain 7711 were suspended in phosphate citrate buffer (40/20 mM) at arange of pH-values from 4.6 to 8.0. ΔPlySs1 was added (110 μg/ml) andOD600 was measured over 60 min (horizontal axis) at 37° C. The verticalaxis represents the treated/untreated OD600-ratio at each timepoint. Foreach pH-value, the curve depicts the running average of 3 independentexperiments. Overall, activity was maximal at the upper end of thebuffering range. (B) Here, bis-tris-propane (40 mM) was employed as thebuffering agent with a pH-range from 7.0 to 9.7; ΔPlySs1 was again addedto 110 μg/ml. Each curve depicts the running average of 3 experiments.Maximal activity was observed at pH=9.0, although the quantitativedegree of OD-decline was, in general, less than in phosphate-citrate.

FIG. 12 depicts ΔPLySS1 NaCl dependence. S. suis 7711 cells weresuspended in phosphate-citrate buffer pH=7.8 (40/20 mM). NaCl was addedto the above concentrations, followed by ΔPlySs1 at 110 μg/ml. Opticaldensity at 600 nm was observed over 60 min at 37° C. In this figure, thevertical axis represents the treated/untreated OD₆₀₀-ratio for each NaClconcentration, averaged over 3 independent experiments.

FIGS. 13A and 13B provides assessment of ΔPlySs1 DTT and EDTAsusceptibility. (A) ΔPlySs1 was pre-incubated for 1 hr with 5 mM DTT (alarge molar excess) prior to addition to 7711 cells; activity wasunchanged. (B) Here, various concentrations of EDTA were included in thebuffered suspension of cells prior to addition of ΔPlySs1 (110 μg/mllysin). For both images, the vertical axis represents thetreated/untreated OD₆₀₀-ratio for each condition, averaged over 3independent experiments.

FIGS. 14A and 14B shows ΔPlySs1 temperature stability. (A) A ΔPlySs1stock solution was held at each of the above temperature for 30 minutes,followed by addition to 7711 cells (270 μg/ml final enzymeconcentration, final temperature=37° C., ideal buffering conditions).The curves in this image represent running averages of 3 individualexperiments. In each case, complete loss of activity was observedbetween the 45° C. and 50° C. samples. The 3 hottest samples show aslightly higher OD₆₀₀ reading than the untreated control due toflocculation of ΔPlySs1 upon denaturation. (B) The above experiment wasrepeated, but with 6 hours of heat-treatment prior to the assay. At thislonger incubation time, the 45° C. sample showed some loss of activity,though not complete. The 40° C. sample maintained essentially nativeactivity.

FIGS. 15A and 15B (A) PlySs2 has acute lytic activity against S. suisstrain 7997 at, or above 8 ug/mL. (B) Activity of PlySs2 assessed invitro against S. suis strain S735.

FIG. 16A-16D provides PlySs2 activity against different species andstrains. S. suis 7997 was used as a positive control for each test. A.PlySs2 activity against strains of S. suis. B. PlySs2 activity againstdifferent species of bacteria and 2 strains of S. suis. C. Streptococciand staphylococci sensitivity to PlySs2. D. Various species tested forsusceptibility to PlySs2 treatment.

FIGS. 17A and 17B shows PlySs2 activity against multiple species,serotypes, and strains of bacteria. In each instance theTreated/Untreated OD₆₀₀ is depicted in a bar graph. The bars of S.aureus strains are colored red; bars corresponding to S. suis strainsare orange. The bars of bacteria Listeria and other bacteria of interestare shown in purple.

FIG. 18. PlySS2 was tested by standard MIC analysis for its ability tokill strains of staphylococci. Included in the testing were resistantstaphylococci such as Vancomycin resistant (VRSA), Vancomycinintermediate (VISA) and methicillin resistant (MRSA) staphylococci. Thethree VRSA strains tested represent half of all known isolates.

FIG. 19 provides ΔPlySs1 bacteriolytic activity. Depicted here areOD-drop curves for three strains of S. suis: 7711, the serotype 7 strainfrom which PlySs1 was originally cloned (i.e. the host strain); S735, aserotype 2 isolate that is the type-strain for the species; and 7997, avirulent serotype 9 strain. Bacteria were suspended in 20 mM phosphatebuffer pH 7.8, 2 mM EDTA (defined as optimal conditions). ΔPlySs1 wasadded to the cells at a range of concentrations (indicated by theinset). For each sample, optical density at 600 nm (vertical axis) wasmeasured over the course of an hour (horizontal axis) at 37□C. In thisimage, all curves represent running averages of 3 or 4 independentexperiments.

FIG. 20 shows ΔPlySs1 growth inhibition of S. suis 7711. ΔPlySs1 wasadded at the above final concentrations to a dilute suspension of S.suis strain 7711 in BHI broth. The optical density of each sample wasmeasured continuously overnight in 96-well plate format. Overall,bacterial growth was delayed in a dose-dependent manner. However, forenzyme-concentrations that were sufficient to induce lysis in bufferedsolutions (130 and 50 μg/ml), the effect was quite minimal here.Moreover, none of the above ΔPlySs1 concentrations inhibited growthoutright-hence, a MIC could not be assigned. For all of the treatedsamples, one will note that the final optical densities are actuallyhigher than that of the untreated sample. This is an artifact of theaccumulation of aggregated bacterial debris that occurred in thepresence of lytic enzyme.

FIG. 21 provides a ΔPlySs1 bacterial strain panel. The informationprovided in FIG. 19 and Tables 3 and 4 is summarized graphically for twoPlySs1 concentrations, 130 μg/ml and 32.5 μg/ml. In the image, strainsof S. suis are denoted with double red asterisks and non-suisstreptococci are denoted with single black asterisks. The opticaldensity response (treated-versus-untreated OD₆₀₀ ratio) after 1 hr isshown. The reader is referred to Table 3 for the serotype definitions ofthe S. suis strains.

FIG. 22 provides CFU killing assay results of PlySs2 bacteriolyticactivity against S. suis strain 7997 and S735.

FIG. 23 depicts the results of an S. aureus and S. pyogenes resistanceassay against PlySS2 compared to antibiotic mupirocin. None of MRSAstrain MW2, MSSA strain 8325, nor S. pyogenes strain 5005 developedresistance against PlySs2 after each was exposed to incrementallyincreasing concentrations of PlySs2. Both MW2 and 8325 developedresistance to the positive control, mupirocin.

FIG. 24 depicts the survival of mice with MRSA bacteremia over 10 days.PlySs2 cleared bacteremia from 95% of mice tested. Of the controls, just5% survived.

FIG. 25 provides PlySs2 activity against different species and strains.Log-phase cultures were exposed to 32 μg/ml PlySs2 for 60 minutes inphosphate buffer. The final OD₆₀₀ of the treated samples were divided bythe final OD₆₀₀ of the untreated samples to generate the normalizedvalues above. Multiple Staphylococci (including, but not limited to:MRSA, MSSA, and VISA), Streptococci, Listeria, Enterococci, and Bacilliwere tested for susceptibility to PlySs2 activity. Escherichia andPseudomonas were tested as Gram-negative controls.

FIG. 26 depicts bactericidal effect of PlySs2 on various strains.Bactericidal effect of 128 μg/ml PlySs2 60 mins post-treatment. Thereduction in CFU counts is presented along a logarithmic scale.Characteristically, PlySs2 had significant activity against MRSA MW2. Ofnote, PlySs2 dramatically reduced S. agalactiae and L. monocytogenes.There was no reduction in number of the negative control E. coli.

FIG. 27 depicts the minimum inhibitory concentration (MIC) of PlySs2 forvarious Gram-positive bacteria. There was a low MIC for MRSA MW2, asexpected, and a higher MIC for S. pyogenes 5005. The MIC of PlySs2correlates to the lytic activity and bactericidal tests. The MIC ofPlySs2 for the negative control E. coli was accordingly immeasurable.

FIG. 28 shows PlySs2 protected mice from death caused by mixed MRSA andS. pyogenes infection. FVB/NJ mice were intraperitoneally injected with5% mucin containing ˜5×10⁵ CFU of MRSA strain MW2, ˜1×10⁷ S. pyogenesstrain 5005, or combination of both bacteria (mixed infection) from theabove inoculums at the same concentrations. Three hours post-infection,mice in all infection groups (A-C), received one intraperitonealinjection of 20 mM phosphate buffer control, 1 mg of ClyS, 1 mg of PlyC,or a combination of 1 mg of ClyS plus 1 mg of PlyC for the mixedinfection. PlySs2 treatments consisted of 1 mg for MRSA infections (A),or 2 mg for S. pyogenes and mixed infections (B-C). Mice were monitoredfor survival over ten days. The results from four independentexperiments were combined and the mice survival data plotted with aKaplan Meier Survival curve.

FIG. 29 depicts activity of PlySs2 and vancomycin against MRSA isolates.

FIG. 30 provides PlySs2 enzymatic domain alignment to ClyS. The CHAPdomains of the streptococcal lysins PlySs2 and PlyC (subunit A, GenBankno. AAP42310) are aligned. Amino-acid identities are indicated withunderlying asterisks and highlighting. The positions of the presumptivecatalytic residues (cysteine and histidine, for which the domain isnamed is named) are indicated with arrows.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions as provided and set out below and in this section.

The terms “S. suis lysin(s)”, “PlySs lysin(s)”, “PlySs1 lysin”,“PlySs1”, “whole PlySs1”, “truncated PlySs1”, “ΔPlySs1”, “PlySs2 lysin”,“PlySs2” and any variants not specifically listed, may be used hereininterchangeably, and as used throughout the present application andclaims refer to proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in FIG. 3 and in FIG. 4 (SEQ ID NOS:1, 2 and/or 3), and the profile of activities set forth herein and inthe Claims. Accordingly, proteins displaying substantially equivalent oraltered activity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained throughsite-directed mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “S. suis lysin(s)”, “PlySs lysin(s)”,“PlySs1 lysin”, “PlySs1”, “whole PlySs1”, “truncated PlySs1”, “ΔPlySs1”,“PlySs2 lysin”, “PlySs2” are intended to include within their scopeproteins specifically recited herein as well as all substantiallyhomologous analogs, fragments or truncations, and allelic variations.

Polypeptides and Lytic Enzymes

A “lytic enzyme” includes any bacterial cell wall lytic enzyme thatkills one or more bacteria under suitable conditions and during arelevant time period. Examples of lytic enzymes include, withoutlimitation, various amidase cell wall lytic enzymes.

A “S. suis lytic enzyme” includes a lytic enzyme that is capable ofkilling at least one or more Streptococcus suis bacteria under suitableconditions and during a relevant time period.

A “bacteriophage lytic enzyme” refers to a lytic enzyme extracted orisolated from a bacteriophage or a synthesized lytic enzyme with asimilar protein structure that maintains a lytic enzyme functionality.

A lytic enzyme is capable of specifically cleaving bonds that arepresent in the peptidoglycan of bacterial cells to disrupt the bacterialcell wall. It is also currently postulated that the bacterial cell wallpeptidoglycan is highly conserved among most bacteria, and cleavage ofonly a few bonds to may disrupt the bacterial cell wall. Thebacteriophage lytic enzyme may be an amidase, although other types ofenzymes are possible. Examples of lytic enzymes that cleave these bondsare various amidases such as muramidases, glucosaminidases,endopeptidases, or N-acetyl-muramoyl-L-alanine amidases. Fischetti et al(1974) reported that the C1 streptococcal phage lysin enzyme was anamidase. Garcia et al (1987, 1990) reported that the Cpl lysin from a S.pneumoniae from a Cp-1 phage was a lysozyme. Caldentey and Bamford(1992) reported that a lytic enzyme from the phi 6 Pseudomonas phage wasan endopeptidase, splitting the peptide bridge formed bymelo-diaminopimilic acid and D-alanine. The E. coli T1 and T6 phagelytic enzymes are amidases as is the lytic enzyme from Listeria phage(ply) (Loessner et al, 1996). There are also other lytic enzymes knownin the art that are capable of cleaving a bacterial cell wall.

A “lytic enzyme genetically coded for by a bacteriophage” includes apolypeptide capable of killing a host bacteria, for instance by havingat least some cell wall lytic activity against the host bacteria. Thepolypeptide may have a sequence that encompasses native sequence lyticenzyme and variants thereof. The polypeptide may be isolated from avariety of sources, such as from a bacteriophage (“phage”), or preparedby recombinant or synthetic methods, such as those described by Garciaet al and also as provided herein. The polypeptide may comprise acholine-binding portion at the carboxyl terminal side and may becharacterized by an enzyme activity capable of cleaving cell wallpeptidoglycan (such as amidase activity to act on amide bonds in thepeptidoglycan) at the amino terminal side. Lytic enzymes have beendescribed which include multiple enzyme activities, for example twoenzymatic domains, such as PlyGBS lysin. Generally speaking, a lyticenzyme may be between 25,000 and 35,000 daltons in molecular weight andcomprise a single polypeptide chain; however, this can vary depending onthe enzyme chain. The molecular weight most conveniently can bedetermined by assay on denaturing sodium dodecyl sulfate gelelectrophoresis and comparison with molecular weight markers.

“A native sequence phage associated lytic enzyme” includes a polypeptidehaving the same amino acid sequence as an enzyme derived from abacteria. Such native sequence enzyme can be isolated or can be producedby recombinant or synthetic means.

The term “native sequence enzyme” encompasses naturally occurring forms(e.g., alternatively spliced or altered forms) and naturally-occurringvariants of the enzyme. In one embodiment of the invention, the nativesequence enzyme is a mature or full-length polypeptide that isgenetically coded for by a gene from a bacteriophage specific forStreptococcus suis. Of course, a number of variants are possible andknown, as acknowledged in publications such as Lopez et al., MicrobialDrug Resistance 3: 199-211 (1997); Garcia et al., Gene 86: 81-88 (1990);Garcia et al., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia etal., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al.,Streptococcal Genetics (J. J. Ferretti and Curtis eds., 1987); Lopez etal., FEMS Microbiol. Lett. 100: 439-448 (1992); Romero et al., J.Bacteriol. 172: 5064-5070 (1990); Ronda et al., Eur. J. Biochem. 164:621-624 (1987) and Sanchez et al., Gene 61: 13-19 (1987). The contentsof each of these references, particularly the sequence listings andassociated text that compares the sequences, including statements aboutsequence homologies, are specifically incorporated by reference in theirentireties.

“A variant sequence lytic enzyme” includes a lytic enzyme characterizedby a polypeptide sequence that is different from that of a lytic enzyme,but retains functional activity. The lytic enzyme can, in someembodiments, be genetically coded for by a bacteriophage specific forStreptococcus suis having a particular amino acid sequence identity withthe lytic enzyme sequence(s) hereof, as provided in FIG. 3 and FIG. 4 orin any of SEQ ID NOS: 1, 2 or 3. For example, in some embodiments, afunctionally active lytic enzyme can kill Streptococcus suis bacteria,and other susceptible bacteria as provided herein, including as shown inTABLE 1 and in FIGS. 9 and 10, by disrupting the cellular wall of thebacteria. An active lytic enzyme may have a 60, 65, 70, 75, 80, 85, 90,95, 97, 98, 99 or 99.5% amino acid sequence identity with the lyticenzyme sequence(s) hereof, as provided in FIG. 3 and FIG. 4 or in any ofSEQ ID NOS: 1, 2 or 3. Such phage associated lytic enzyme variantsinclude, for instance, lytic enzyme polypeptides wherein one or moreamino acid residues are added, or deleted at the N or C terminus of thesequence of the lytic enzyme sequence(s) hereof, as provided in FIG. 3and FIG. 4 or in any of SEQ ID NOS: 1, 2 or 3. In a particular aspect, aphage associated lytic enzyme will have at least about 80% or 85% aminoacid sequence identity with native phage associated lytic enzymesequences, particularly at least about 90% (e.g. 90%) amino acidsequence identity. Most particularly a phage associated lytic enzymevariant will have at least about 95% (e.g. 95%) amino acid sequenceidentity with the native phage associated the lytic enzyme sequence(s)hereof, as provided in FIG. 3 and FIG. 4 or in any of SEQ ID NOS: 1, 2or 3.

“Percent amino acid sequence identity” with respect to the phageassociated lytic enzyme sequences identified is defined herein as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the phage associated lyticenzyme sequence, after aligning the sequences in the same reading frameand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity.

“Percent nucleic acid sequence identity” with respect to the phageassociated lytic enzyme sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the phage associated lytic enzyme sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

To determine the percent identity of two nucleotide or amino acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps may be introduced in the sequence of a first nucleotidesequence). The nucleotides or amino acids at corresponding nucleotide oramino acid positions are then compared. When a position in the firstsequence is occupied by the same nucleotide or amino acid as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions.times.100).

The determination of percent identity between two sequences may beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin et al., Proc. Natl. Acad. Sci. USA,90:5873-5877 (1993). Such an algorithm is incorporated into the NBLASTprogram which may be used to identify sequences having the desiredidentity to nucleotide sequences of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST may be utilized asdescribed in Altschul et al., Nucleic Acids Res, 25:3389-3402 (1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., NBLAST) may be used. See the programsprovided by National Center for Biotechnology Information, NationalLibrary of Medicine, National Institutes of Health. In one embodiment,parameters for sequence comparison may be set at W=12. Parameters mayalso be varied (e.g., W=5 or W=20). The value “W” determines how manycontinuous nucleotides must be identical for the program to identify twosequences as containing regions of identity.

“Polypeptide” includes a polymer molecule comprised of multiple aminoacids joined in a linear manner. A polypeptide can, in some embodiments,correspond to molecules encoded by a polynucleotide sequence which isnaturally occurring. The polypeptide may include conservativesubstitutions where the naturally occurring amino acid is replaced byone having similar properties, where such conservative substitutions donot alter the function of the polypeptide (see, for example, Lewin“Genes V” Oxford University Press Chapter 1, pp. 9-13 1994).

The term “altered lytic enzymes” includes shuffled and/or chimeric lyticenzymes.

Phage lytic enzymes specific for bacteria infected with a specific phagehave been found to effectively and efficiently break down the cell wallof the bacterium in question. The lytic enzyme is believed to lackproteolytic enzymatic activity and is therefore non-destructive tomammalian proteins and tissues when present during the digestion of thebacterial cell wall. As shown by Loeffler et al., “Rapid Killing ofStreptococcus pneumoniae with a Bacteriophage Cell Wall Hydrolase,”Science, 294: 2170-2172 (Dec. 7, 2001), and supplemental materialthereto published online by Science magazine, which are incorporatedherein by reference in their entirety, a purified pneumococcalbacteriophage lytic enzyme, such as Pal, is able to kill variouspneumococci. Loeffler et al. have shown through these experiments thatwithin seconds after contact, the lytic enzyme Pal is able to kill 15clinical stains of S. pneumoniae, including the most frequently isolatedserogroups and penicillin resistant stains, in vitro. Treatment of micewith Pal was also able to eliminate or significantly reduce nasalcarriage of serotype 14 in a dose-dependent manner. Furthermore, becauseit has been found that the action of Pal, like other phage lyticenzymes, but unlike antibiotics, was rather specific for the targetpathogen, it is likely that the normal flora will remain essentiallyintact (M. J. Loessner, G. Wendlinger, S. Scherer, Mol Microbiol 16,1231-41. (1995) incorporated herein by reference). In contrast, lysinpolypeptide of the present invention has a remarkably broad andclinically significant bacterial killing profile. As demonstratedherein, for example, the isolated S. suis lysin PlySs2, is effective inkilling S. suis, and also various other Streptococcus strains, includingGroup B Streptococcus (GBS), Staphylococcal strains, includingStaphylococcus aureus, Enterococcus and Listeria. The lysin of thepresent invention thus demonstrates a breadth of bacterial cell killingunlike any lysin previously reported or contemplated.

A lytic enzyme or polypeptide of the invention may be produced by thebacterial organism after being infected with a particular bacteriophageas either a prophylactic treatment for preventing those who have beenexposed to others who have the symptoms of an infection from gettingsick, or as a therapeutic treatment for those who have already becomeill from the infection. In as much the lysin polypeptide sequences andnucleic acids encoding the lysin polypeptides are provided herein, thelytic enzyme(s)/polypeptide(s) may be preferably produced via theisolated gene for the lytic enzyme from the phage genome, putting thegene into a transfer vector, and cloning said transfer vector into anexpression system, using standard methods of the art, including asexemplified herein. The lytic enzyme(s) or polypeptide(s) may betruncated, chimeric, shuffled or “natural,” and may be in combination.Relevant U.S. Pat. No. 5,604,109 is incorporated herein in its entiretyby reference. An “altered” lytic enzyme can be produced in a number ofways. In a preferred embodiment, a gene for the altered lytic enzymefrom the phage genome is put into a transfer or movable vector,preferably a plasmid, and the plasmid is cloned into an expressionvector or expression system. The expression vector for producing a lysinpolypeptide or enzyme of the invention may be suitable for E. coli,Bacillus, or a number of other suitable bacteria. The vector system mayalso be a cell free expression system. All of these methods ofexpressing a gene or set of genes are known in the art. The lytic enzymemay also be created by infecting Streptococcus suis with a bacteriophagespecific for Streptococcus suis, wherein said at least one lytic enzymeexclusively lyses the cell wall of said Streptococcus suis having atmost minimal effects on other, for example natural or commensal,bacterial flora present.

A “chimeric protein” or “fusion protein” comprises all or (preferably abiologically active) part of a polypeptide of the invention operablylinked to a heterologous polypeptide. Chimeric proteins or peptides areproduced, for example, by combining two or more proteins having two ormore active sites. Chimeric protein and peptides can act independentlyon the same or different molecules, and hence have a potential to treattwo or more different bacterial infections at the same time. Chimericproteins and peptides also may be used to treat a bacterial infection bycleaving the cell wall in more than one location, thus potentiallyproviding more rapid or effective (or synergistic) killing from a singlelysin molecule or chimeric peptide.

A “heterologous” region of a DNA construct or peptide construct is anidentifiable segment of DNA within a larger DNA molecule or peptidewithin a larger peptide molecule that is not found in association withthe larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA or peptide as defined herein.

The term “operably linked” means that the polypeptide of the disclosureand the heterologous polypeptide are fused in-frame. The heterologouspolypeptide can be fused to the N-terminus or C-terminus of thepolypeptide of the disclosure. Chimeric proteins are producedenzymatically by chemical synthesis, or by recombinant DNA technology. Anumber of chimeric lytic enzymes have been produced and studied. GeneE-L, a chimeric lysis constructed from bacteriophages phi X174 and MS2lysis proteins E and L, respectively, was subjected to internaldeletions to create a series of new E-L clones with altered lysis orkilling properties. The lytic activities of the parental genes E, L,E-L, and the internal truncated forms of E-L were investigated in thisstudy to characterize the different lysis mechanism, based ondifferences in the architecture of the different membranes spanningdomains. Electron microscopy and release of marker enzymes for thecytoplasmic and periplasmic spaces revealed that two different lysismechanisms can be distinguished depending on penetration of the proteinsof either the inner membrane or the inner and outer membranes of the E.coli (FEMS Microbiol. Lett. (1998) 164(1):159-67 (incorporated herein byreference). One example of a useful fusion protein is a GST fusionprotein in which the polypeptide of the disclosure is fused to theC-terminus of a GST sequence. Such a chimeric protein can facilitate thepurification of a recombinant polypeptide of the disclosure.

In another embodiment, the chimeric protein or peptide contains aheterologous signal sequence at its N-terminus. For example, the nativesignal sequence of a polypeptide of the disclosure can be removed andreplaced with a signal sequence from another protein. For example, thegp67 secretory sequence of the baculovirus envelope protein can be usedas a heterologous signal sequence (Current Protocols in MolecularBiology, Ausubel et al., eds., John Wiley & Sons, 1992, incorporatedherein by reference). Other examples of eukaryotic heterologous signalsequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Sambrook et al., supra) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

The fusion protein may combine a lysin polypeptide with a protein orpolypeptide of having a different capability, or providing an additionalcapability or added character to the lysin polypeptide. The fusionprotein may be an immunoglobulin fusion protein in which all or part ofa polypeptide of the disclosure is fused to sequences derived from amember of the immunoglobulin protein family. The immunoglobulin may bean antibody, for example an antibody directed to a surface protein orepitope of a susceptible or target bacteria. An immunoglobulin fusionprotein can be incorporated into a pharmaceutical composition andadministered to a subject to inhibit an interaction between a ligand(soluble or membrane-bound) and a protein on the surface of a cell(receptor), to thereby suppress signal transduction in vivo. Theimmunoglobulin fusion protein can alter bioavailability of a cognateligand of a polypeptide of the disclosure. Inhibition of ligand/receptorinteraction may be useful therapeutically, both for treatingbacterial-associated diseases and disorders for modulating (i.e.promoting or inhibiting) cell survival. Moreover, an immunoglobulinfusion protein of the disclosure can be used as an immunogen to produceantibodies directed against a polypeptide of the disclosure in asubject, to purify ligands and in screening assays to identify moleculeswhich inhibit the interaction of receptors with ligands. Chimeric andfusion proteins and peptides of the disclosure can be produced bystandard recombinant DNA techniques.

The fusion gene can be synthesized by conventional techniques, includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments whichsubsequently can be annealed and reamplified to generate a chimeric genesequence (see, i.e., Ausubel et al., supra). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(i.e., a GST polypeptide). A nucleic acid encoding a polypeptide of theinvention can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the polypeptide of the invention.

As used herein, shuffled proteins or peptides, gene products, orpeptides for more than one related phage protein or protein peptidefragments have been randomly cleaved and reassembled into a more activeor specific protein. Shuffled oligonucleotides, peptides or peptidefragment molecules are selected or screened to identify a moleculehaving a desired functional property. This method is described, forexample, in Stemmer, U.S. Pat. No. 6,132,970. (Method of shufflingpolynucleotides); Kauffman, U.S. Pat. No. 5,976,862 (Evolution viaCondon-based Synthesis) and Huse, U.S. Pat. No. 5,808,022 (Direct CodonSynthesis). The contents of these patents are incorporated herein byreference. Shuffling can be used to create a protein that is moreactive, for instance up to 10 to 100 fold more active than the templateprotein. The template protein is selected among different varieties oflysin proteins. The shuffled protein or peptides constitute, forexample, one or more binding domains and one or more catalytic domains.Each binding or catalytic domain is derived from the same or a differentphage or phage protein. The shuffled domains are either oligonucleotidebased molecules, as gene or gene products, that either alone or incombination with other genes or gene products are translatable into apeptide fragment, or they are peptide based molecules. Gene fragmentsinclude any molecules of DNA, RNA, DNA-RNA hybrid, antisense RNA,Ribozymes, ESTs, SNIPs and other oligonucleotide-based molecules thateither alone or in combination with other molecules produce anoligonucleotide molecule capable or incapable of translation into apeptide.

The modified or altered form of the protein or peptides and peptidefragments, as disclosed herein, includes protein or peptides and peptidefragments that are chemically synthesized or prepared by recombinant DNAtechniques, or both. These techniques include, for example,chimerization and shuffling. When the protein or peptide is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of the protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than the polypeptide of interest.

A signal sequence of a polypeptide can facilitate transmembrane movementof the protein and peptides and peptide fragments of the disclosure toand from mucous membranes, as well as by facilitating secretion andisolation of the secreted protein or other proteins of interest. Signalsequences are typically characterized by a core of hydrophobic aminoacids which are generally cleaved from the mature protein duringsecretion in one or more cleavage events. Such signal peptides containprocessing sites that allow cleavage of the signal sequence from themature proteins as they pass through the secretory pathway. Thus, thedisclosure can pertain to the described polypeptides having a signalsequence, as well as to the signal sequence itself and to thepolypeptide in the absence of the signal sequence (i.e., the cleavageproducts). A nucleic acid sequence encoding a signal sequence of thedisclosure can be operably linked in an expression vector to a proteinof interest, such as a protein which is ordinarily not secreted or isotherwise difficult to isolate. The signal sequence directs secretion ofthe protein, such as from an eukaryotic host into which the expressionvector is transformed, and the signal sequence is subsequently orconcurrently cleaved. The protein can then be readily purified from theextracellular medium by art-recognized methods. Alternatively, thesignal sequence can be linked to a protein of interest using a sequencewhich facilitates purification, such as with a GST domain.

The present invention also pertains to other variants of thepolypeptides of the invention. Such variants may have an altered aminoacid sequence which can function as either agonists (mimetics) or asantagonists. Variants can be generated by mutagenesis, i.e., discretepoint mutation or truncation. An agonist can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the protein. An antagonist of a protein can inhibitone or more of the activities of the naturally occurring form of theprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes theprotein of interest. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theprotein. Variants of a protein of the disclosure which function aseither agonists (mimetics) or as antagonists can be identified byscreening combinatorial libraries of mutants, i.e., truncation mutants,of the protein of the disclosure for agonist or antagonist activity. Inone embodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (i.e., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the disclosurefrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, i.e., Narang(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477, all herein incorporated by reference).

In addition, libraries of fragments of the coding sequence of apolypeptide of the disclosure can be used to generate a variegatedpopulation of polypeptides for screening and subsequent selection ofvariants, active fragments or truncations. For example, a library ofcoding sequence fragments can be generated by treating a double strandedPCR fragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the protein of interest. Severaltechniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the disclosure (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331) immunologically active portions of a protein or peptidefragment include regions that bind to antibodies that recognize thephage enzyme. In this context, the smallest portion of a protein (ornucleic acid that encodes the protein) according to embodiments is anepitope that is recognizable as specific for the phage that makes thelysin protein. Accordingly, the smallest polypeptide (and associatednucleic acid that encodes the polypeptide) that can be expected to binda target or receptor, such as an antibody, and is useful for someembodiments may be 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 75, 85, or 100 amino acids long. Although small sequences asshort as 8, 9, 10, 11, 12 or 15 amino acids long reliably compriseenough structure to act as targets or epitopes, shorter sequences of 5,6, or 7 amino acids long can exhibit target or epitopic structure insome conditions and have value in an embodiment. Thus, the smallestportion of the protein(s) or lysin polypeptides provided herein,including as set out in FIGS. 3 and 4 and in SEQ ID NOS: 1, 2 and/or 3,includes polypeptides as small as 5, 6, 7, 8, 9, 10, 12, 14 or 16 aminoacids long.

Biologically active portions of a protein or peptide fragment of theembodiments, as described herein, include polypeptides comprising aminoacid sequences sufficiently identical to or derived from the amino acidsequence of the phage protein of the disclosure, which include feweramino acids than the full length protein of the phage protein andexhibit at least one activity of the corresponding full-length protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the corresponding protein. A biologicallyactive portion of a protein or protein fragment of the disclosure can bea polypeptide which is, for example, 10, 25, 50, 100 less or more aminoacids in length. Moreover, other biologically active portions, in whichother regions of the protein are deleted, or added can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of the native form of a polypeptide of the embodiments.

Homologous proteins and nucleic acids can be prepared that sharefunctionality with such small proteins and/or nucleic acids (or proteinand/or nucleic acid regions of larger molecules) as will be appreciatedby a skilled artisan. Such small molecules and short regions of largermolecules that may be homologous specifically are intended asembodiments. Preferably the homology of such valuable regions is atleast 50%, 65%, 75%, 80%, 85%, and preferably at least 90%, 95%, 97%,98%, or at least 99% compared to the lysin polypeptides provided herein,including as set out in FIGS. 3 and 4 and in SEQ ID NOS: 1, 2 and/or 3.These percent homology values do not include alterations due toconservative amino acid substitutions.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, atleast about 85%, and preferably at least about 90 or 95%) are identical,or represent conservative substitutions. The sequences of comparablelysins, such as comparable PlySs2 lysins, or comparable PlySs1 lysins,are substantially homologous when one or more, or several, or up to 10%,or up to 15%, or up to 20% of the amino acids of the lysin polypeptideare substituted with a similar or conservative amino acid substitution,and wherein the comparable lysins have the profile of activities,anti-bacterial effects, and/or bacterial specificities of a lysin, suchas the PlySs2 and/or PlySs1 lysins, disclosed herein.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfuctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

Mutations can be made in the amino acid sequences, or in the nucleicacid sequences encoding the polypeptides and lysins herein, including inthe lysin sequences set out in FIG. 3 or in FIG. 4, or in activefragments or truncations thereof, such that a particular codon ischanged to a codon which codes for a different amino acid, an amino acidis substituted for another amino acid, or one or more amino acids aredeleted. Such a mutation is generally made by making the fewest aminoacid or nucleotide changes possible. A substitution mutation of thissort can be made to change an amino acid in the resulting protein in anon-conservative manner (for example, by changing the codon from anamino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to another grouping)or in a conservative manner (for example, by changing the codon from anamino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to the same grouping).Such a conservative change generally leads to less change in thestructure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein.

Thus, one of skill in the art, based on a review of the sequence of thePlySs1 and PlySs2 lysin polypeptides provided herein and on theirknowledge and the public information available for other lysinpolypeptides, can make amino acid changes or substitutions in the lysinpolypeptide sequence. Amino acid changes can be made to replace orsubstitute one or more, one or a few, one or several, one to five, oneto ten, or such other number of amino acids in the sequence of thelysin(s) provided herein to generate mutants or variants thereof. Suchmutants or variants thereof may be predicted for function or tested forfunction or capability for killing bacteria, including Staphylococcal,Streptococcal, Listeria, or Enterococcal bacteria, and/or for havingcomparable activity to the lysin(s) provided herein. Thus, changes canbe made to the sequence of PlySs2, for example, by modifying the aminoacid sequence as set out in FIG. 4 hereof, and mutants or variantshaving a change in sequence can be tested using the assays and methodsdescribed and exemplified herein, including in the examples. One ofskill in the art, on the basis of the domain structure of the lysin(s)hereof can predict one or more, one or several amino acids suitable forsubstitution or replacement and/or one or more amino acids which are notsuitable for substitution or replacement, including reasonableconservative or non-conservative substitutions.

In this regard, and with exemplary reference to PlySs2 lysin it ispointed out that, although the PlySs2 polypeptide lysin represents adivergent class of prophage lytic enzyme, the lysin comprises anN-terminal CHAP domain (cysteine-histidine amidohydrolase/peptidase) anda C-terminal SH3-type 5 domain as depicted in FIG. 4. The domains aredepicted in the amino acid sequence in distinct shaded color regions,with the CHAP domain corresponding to the first shaded amino acidsequence region starting with LNN . . . and the SH3-type 5 domaincorresponding to the second shaded region starting with RSY . . . CHAPdomains are included in several previously characterized streptococcaland staphylococcal phage lysins. Thus, one of skill in the art canreasonably make and test substitutions or replacements to the CHAPdomain and/or the SH-3 domain of PlySs2. Sequence comparisons to theGenbank database can be made with either or both of the CHAP and/or SH-3domain sequences or with the PlySs2 lysin full amino acid sequence, forinstance, to identify amino acids for substitution. In FIG. 30, the CHAPdomain of PlySs2 is aligned with that of the well-characterizedstreptococcal PlyC lysin, demonstrating conserved catalytic residues,but only a modest level of identity overall (28% sequence identity). InFIG. 30 the conserved cysteine and histidine amino acid sequences in theCHAP domain are shown with an arrow. It is reasonable to predict, forexample, that the conserved cysteine and histidine residues should bemaintained in a mutant or variant of PlySs2 so as to maintain activityor capability. It is notable that a mutant or variant having an alaninereplaced for valine at valine amino acid 19 in the PlySs2 sequence ofFIG. 4 and SEQ ID NO: 3 is active and capable of killing gram positivebacteria in a manner similar to and as effective as the FIG. 4 and SEQID NO:3 lysin.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,Tryptophan, Methionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0) Lysine, Arginine,Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Exemplary and preferred conservative amino acid substitutions includeany of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L)for valine (V) and vice versa; serine (S) for threonine (T) and viceversa; isoleucine (I) for valine (V) and vice versa; lysine (K) forglutamine (Q) and vice versa; isoleucine (I) for methionine (M) and viceversa; serine (S) for asparagine (N) and vice versa; leucine (L) formethionine (M) and vice versa; lysine (L) for glutamic acid (E) and viceversa; alanine (A) for serine (S) and vice versa; tyrosine (Y) forphenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid(D) and vice versa; leucine (L) for isoleucine (I) and vice versa;lysine (K) for arginine (R) and vice versa.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

A polypeptide or epitope as described herein may be used to generate anantibody and also can be used to detect binding to the lysin or tomolecules that recognize the lysin protein. Another embodiment is amolecule such as an antibody or other specific binder that may becreated through use of an epitope such as by regular immunization or bya phase display approach where an epitope can be used to screen alibrary if potential binders. Such molecules recognize one or moreepitopes of lysin protein or a nucleic acid that encodes lysin protein.An antibody that recognizes an epitope may be a monoclonal antibody, ahumanized antibody, or a portion of an antibody protein. Desirably themolecule that recognizes an epitope has a specific binding for thatepitope which is at least 10 times as strong as the molecule has forserum albumin. Specific binding can be measured as affinity (Km). Moredesirably the specific binding is at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, or even higher than that for serum albumin under the sameconditions.

In a desirable embodiment the antibody or antibody fragment is in a formuseful for detecting the presence of the lysin protein or, alternativelydetecting the presence of a bacteria susceptible to the lysin protein.In a further embodiment the antibody may be attached or otherwiseassociated with the lysin polypeptide of the invention, for example in achimeric or fusion protein, and may serve to direct the lysin to abacterial cell or strain of interest or target. Alternatively, the lysinpolypeptide may serve to direct the antibody or act in conjunction withthe antibody, for example in lysing the bacterial cell wall fully orpartially, so that the antibody may specifically bind to its epitope atthe surface or under the surface on or in the bacteria. For example, alysin of the invention may be attached to an anti-Streptococcal antibodyand direct the antibody to its epitope.

A variety of forms and methods for antibody synthesis are known as willbe appreciated by a skilled artisan. The antibody may be conjugated(covalently complexed) with a reporter molecule or atom such as a fluor,an enzyme that creates an optical signal, a chemilumiphore, amicroparticle, or a radioactive atom. The antibody or antibody fragmentmay be synthesized in vivo, after immunization of an animal, forexample, the antibody or antibody fragment may be synthesized via cellculture after genetic recombination. The antibody or antibody fragmentmay be prepared by a combination of cell synthesis and chemicalmodification.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567. The term“antibody” describes an immunoglobulin whether natural or partly orwholly synthetically produced. The term also covers any polypeptide orprotein having a binding domain which is, or is homologous to, anantibody binding domain. CDR grafted antibodies are also contemplated bythis term. An “antibody” is any immunoglobulin, including antibodies andfragments thereof, that binds a specific epitope. The term encompassespolyclonal, monoclonal, and chimeric antibodies, the last mentioneddescribed in further detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.The term “antibody(ies)” includes a wild type immunoglobulin (Ig)molecule, generally comprising four full length polypeptide chains, twoheavy (H) chains and two light (L) chains, or an equivalent Ig homologuethereof (e.g., a camelid nanobody, which comprises only a heavy chain);including full length functional mutants, variants, or derivativesthereof, which retain the essential epitope binding features of an Igmolecule, and including dual specific, bispecific, multispecific, anddual variable domain antibodies; Immunoglobulin molecules can be of anyclass (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2). Also included within the meaning ofthe term “antibody” are any “antibody fragment”.

An “antibody fragment” means a molecule comprising at least onepolypeptide chain that is not full length, including (i) a Fab fragment,which is a monovalent fragment consisting of the variable light (VL),variable heavy (VH), constant light (CL) and constant heavy 1 (CH1)domains; (ii) a F(ab′)2 fragment, which is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a heavy chain portion of an Fab (Fd) fragment, whichconsists of the VH and CH1 domains; (iv) a variable fragment (Fv)fragment, which consists of the VL and VH domains of a single arm of anantibody, (v) a domain antibody (dAb) fragment, which comprises a singlevariable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) acamelid antibody; (vii) an isolated complementarity determining region(CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VLdomain are linked by a peptide linker which allows the two domains toassociate to form an antigen binding site (Bird et al, Science, 242,423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) adiabody, which is a bivalent, bispecific antibody in which VH and VLdomains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with the complementaritydomains of another chain and creating two antigen binding sites(WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448,(1993)); and (x) a linear antibody, which comprises a pair of tandem Fvsegments (VH-CH1-VH-CH1) which, together with complementarity lightchain polypeptides, form a pair of antigen binding regions; (xi)multivalent antibody fragments (scFv dimers, trimers and/or tetramers(Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); and (xii)other non-full length portions of heavy and/or light chains, or mutants,variants, or derivatives thereof, alone or in any combination.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving a binding domain with the required specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin-binding domain, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an immunoglobulin bindingdomain, or equivalent, fused to another polypeptide are thereforeincluded. Cloning and expression of chimeric antibodies are described inEP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of light chain or heavy and light chain variable andhypervariable regions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule. Exemplaryantibody molecules are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules and those portions of an immunoglobulinmolecule that contains the paratope, including those portions known inthe art as Fab, Fab′, F(ab′)₂ and F(v), which portions are preferred foruse in the therapeutic methods described herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show significant binding tomolecules other than its specific binding partner(s). The term is alsoapplicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “comprise” generally used in the sense of include, that is tosay permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention hereof, those of skill in the art will appreciate thatminor modifications to the N- or C-terminal of the peptide may howeverbe contemplated, such as the chemical modification of the terminal toadd a protecting group or the like, e.g. the amidation of theC-terminus.

The term “isolated” refers to the state in which the lysinpolypeptide(s) of the invention, or nucleic acid encoding suchpolypeptides will be, in accordance with the present invention.Polypeptides and nucleic acid will be free or substantially free ofmaterial with which they are naturally associated such as otherpolypeptides or nucleic acids with which they are found in their naturalenvironment, or the environment in which they are prepared (e.g. cellculture) when such preparation is by recombinant DNA technologypractised in vitro or in vivo. Polypeptides and nucleic acid may beformulated with diluents or adjuvants and still for practical purposesbe isolated—for example the polypeptides will normally be mixed withpolymers or mucoadhesives or other carriers, or will be mixed withpharmaceutically acceptable carriers or diluents, when used in diagnosisor therapy.

Nucleic Acids

Nucleic acids capable of encoding the S. suis lysin polypeptide(s) ofthe invention are provided herein and constitute an aspect of theinvention. Representative nucleic acid sequences in this context arepolynucleotide sequences coding for the polypeptide of any of FIGS. 3and 4, the polypeptides of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, andsequences that hybridize, under stringent conditions, with complementarysequences of the DNA of the FIG. 3 or 4 sequence(s). Further variants ofthese sequences and sequences of nucleic acids that hybridize with thoseshown in the figures also are contemplated for use in production oflysing enzymes according to the disclosure, including natural variantsthat may be obtained. A large variety of isolated nucleic acid sequencesor cDNA sequences that encode phage associated lysing enzymes andpartial sequences that hybridize with such gene sequences are useful forrecombinant production of the lysin enzyme(s) or polypeptide(s) of theinvention.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

Many of the herein contemplated variant DNA molecules include thosecreated by standard DNA mutagenesis techniques, such as M13 primermutagenesis. Details of these techniques are provided in Sambrook et al.(1989) In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y. (incorporated herein by reference). By the use of such techniques,variants may be created which differ in minor ways from those disclosed.DNA molecules and nucleotide sequences which are derivatives of thosespecifically disclosed herein and which differ from those disclosed bythe deletion, addition or substitution of nucleotides while stillencoding a protein which possesses the functional characteristic of thelysin polypeptide(s) are contemplated by the disclosure. Also includedare small DNA molecules which are derived from the disclosed DNAmolecules. Such small DNA molecules include oligonucleotides suitablefor use as hybridization probes or polymerase chain reaction (PCR)primers. As such, these small DNA molecules will comprise at least asegment of a lytic enzyme genetically coded for by a bacteriophage ofStaphylococcus suis and, for the purposes of PCR, will comprise at leasta 10-15 nucleotide sequence and, more preferably, a 15-30 nucleotidesequence of the gene. DNA molecules and nucleotide sequences which arederived from the disclosed DNA molecules as described above may also bedefined as DNA sequences which hybridize under stringent conditions tothe DNA sequences disclosed, or fragments thereof.

Hybridization conditions corresponding to particular degrees ofstringency vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the sodium ion concentration) of the hybridization bufferwill determine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (1989), In MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., chapters 9 and11 (herein incorporated by reference).

An example of such calculation is as follows. A hybridization experimentmay be performed by hybridization of a DNA molecule (for example, anatural variation of the lytic enzyme genetically coded for by abacteriophage specific for Bacillus anthracis) to a target DNA molecule.A target DNA may be, for example, the corresponding cDNA which has beenelectrophoresed in an agarose gel and transferred to a nitrocellulosemembrane by Southern blotting (Southern (1975). J. Mol. Biol. 98:503), atechnique well known in the art and described in Sambrook et al. (1989)In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.(incorporated herein by reference). Hybridization with a target probelabeled with isotopic P³² labeled-dCTP is carried out in a solution ofhigh ionic strength such as 6 times SSC at a temperature that is 20-25degrees Celsius below the melting temperature, Tm (described infra). Forsuch Southern hybridization experiments where the target DNA molecule onthe Southern blot contains 10 ng of DNA or more, hybridization iscarried out for 6-8 hours using 1-2 ng/ml radiolabeled probe (ofspecific activity equal to 109 CPM/mug or greater). Followinghybridization, the nitrocellulose filter is washed to remove backgroundhybridization. The washing conditions are as stringent as possible toremove background hybridization while retaining a specific hybridizationsignal. The term “Tm” represents the temperature above which, under theprevailing ionic conditions, the radiolabeled probe molecule will nothybridize to its target DNA molecule. The Tm of such a hybrid moleculemay be estimated from the following equation: T_(m)=81.5° C.−16.6(log 10of sodium ion concentration)+0.41(% G+C)−0.63(% formamide)−(600/1) wherel=the length of the hybrid in base pairs. This equation is valid forconcentrations of sodium ion in the range of 0.01M to 0.4M, and it isless accurate for calculations of Tm in solutions of higher sodium ionconcentration (Bolton and McCarthy (1962). Proc. Natl. Acad. Sci. USA48:1390) (incorporated herein by reference). The equation also is validfor DNA having G+C contents within 30% to 75%, and also applies tohybrids greater than 100 nucleotides in length. The behavior ofoligonucleotide probes is described in detail in Ch. 11 of Sambrook etal. (1989), In Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y. (incorporated herein by reference). The preferredexemplified conditions described here are particularly contemplated foruse in selecting variations of the lytic gene.

In preferred embodiments of the present disclosure, stringent conditionsmay be defined as those under which DNA molecules with more than 25%sequence variation (also termed “mismatch”) will not hybridize. In amore preferred embodiment, stringent conditions are those under whichDNA molecules with more than 15% mismatch will not hybridize, and morepreferably still, stringent conditions are those under which DNAsequences with more than 10% mismatch will not hybridize. Preferably,stringent conditions are those under which DNA sequences with more than6% mismatch will not hybridize.

The degeneracy of the genetic code further widens the scope of theembodiments as it enables major variations in the nucleotide sequence ofa DNA molecule while maintaining the amino acid sequence of the encodedprotein. For example, a representative amino acid residue is alanine.This may be encoded in the cDNA by the nucleotide codon triplet GCT.Because of the degeneracy of the genetic code, three other nucleotidecodon triplets—GCT, GCC and GCA—also code for alanine. Thus, thenucleotide sequence of the gene could be changed at this position to anyof these three codons without affecting the amino acid composition ofthe encoded protein or the characteristics of the protein. The geneticcode and variations in nucleotide codons for particular amino acids arewell known to the skilled artisan. Based upon the degeneracy of thegenetic code, variant DNA molecules may be derived from the cDNAmolecules disclosed herein using standard DNA mutagenesis techniques asdescribed above, or by synthesis of DNA sequences. DNA sequences whichdo not hybridize under stringent conditions to the cDNA sequencesdisclosed by virtue of sequence variation based on the degeneracy of thegenetic code are herein comprehended by this disclosure.

Thus, it should be appreciated that also within the scope of the presentinvention are DNA sequences encoding a lysin of the present invention,including PlySs2 and PlySs1, which sequences code for a polypeptidehaving the same amino acid sequence as provided in FIG. 3 or 4 or in SEQID NO:1, 2 or 3, but which are degenerate thereto or are degenerate tothe exemplary nucleic acids sequences provided in FIG. 3 or 4. By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L)UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (Ile or I)AUU or AUC or AUA Methionine (Met or M) AUG Valine (Val or V)GUU or GUC of GUA or GUG Serine (Ser or S) UCU or UCC or UCA or UCGor AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCGThreonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Ala or A)GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UACHistidine (His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAGAsparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAGAspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAGCysteine (Cys or C) UGU or UGC Arginine (Arg or R)CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G)GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codon UAA(ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

One skilled in the art will recognize that the DNA mutagenesistechniques described here and known in the art can produce a widevariety of DNA molecules that code for a bacteriophage lysin ofStreptococcus suis yet that maintain the essential characteristics ofthe lytic polypeptides described and provided herein. Newly derivedproteins may also be selected in order to obtain variations on thecharacteristic of the lytic polypeptide(s), as will be more fullydescribed below. Such derivatives include those with variations in aminoacid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation may bepredetermined, the mutation per se does not need to be predetermined.For example, in order to optimize the performance of a mutation at agiven site, random mutagenesis may be conducted at the target codon orregion and the expressed protein variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence asdescribed above are well known.

Amino acid substitutions are typically of single residues, or can be ofone or more, one or a few, one, two, three, four, five, six or sevenresidues; insertions usually will be on the order of about from 1 to 10amino acid residues; and deletions will range about from 1 to 30residues. Deletions or insertions may be in single form, but preferablyare made in adjacent pairs, i.e., a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. Obviously, themutations that are made in the DNA encoding the protein must not placethe sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure (EP75,444A).

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Such substitutions may be made so as to generate nosignificant effect on the protein characteristics or when it is desiredto finely modulate the characteristics of the protein. Amino acids whichmay be substituted for an original amino acid in a protein and which areregarded as conservative substitutions are described above and will berecognized by one of skill in the art.

Substantial changes in function or immunological identity may be made byselecting substitutions that are less conservative, for example byselecting residues that differ more significantly in their effect onmaintaining: (a) the structure of the polypeptide backbone in the areaof the substitution, for example, as a sheet or helical conformation;(b) the charge or hydrophobicity of the molecule at the target site; or(c) the bulk of the side chain. The substitutions which in general areexpected to produce the greatest changes in protein properties will bethose in which: (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histadyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The effects of these amino acid substitutions or deletions or additionsmay be assessed for derivatives or variants of the lytic polypeptide(s)by analyzing the ability of the derivative or variant proteins to lyseor kill susceptible bacteria, or to complement the sensitivity to DNAcross-linking agents exhibited by phages in infected bacteria hosts.These assays may be performed by transfecting DNA molecules encoding thederivative or variant proteins into the bacteria as described above orby incubating bacteria with expressed proteins from hosts transfectedwith the DNA molecules encoding the derivative or variant proteins.

While the site for introducing an amino acid sequence variation can bepredetermined, the mutation per se does not need to be predetermined.For example, in order to optimize the performance of a mutation at agiven site, random mutagenesis may be conducted at the target codon orregion and the expressed protein variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence asdescribed above are well known.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host. Such operative linking ofa DNA sequence of this invention to an expression control sequence, ofcourse, includes, if not already part of the DNA sequence, the provisionof an initiation codon, ATG, in the correct reading frame upstream ofthe DNA sequence. A wide variety of host/expression vector combinationsmay be employed in expressing the DNA sequences of this invention.Useful expression vectors, for example, may consist of segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Suitablevectors include derivatives of SV40 and known bacterial plasmids, e.g.,E. coli plasmids colE1, pCR1, pBR322, pMB9 and their derivatives,plasmids such as RP4; phage DNAS, e.g., the numerous derivatives ofphage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentoussingle stranded phage DNA; yeast plasmids such as the 2□ plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast □-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention.

Libraries of fragments of the coding sequence of a polypeptide can beused to generate a variegated population of polypeptides for screeningand subsequent selection of variants. For example, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Compositions

Therapeutic or pharmaceutical compositions comprising the lyticenzyme(s)/polypeptide(s) of the invention are provided in accordancewith the invention, as well as related methods of use and methods ofmanufacture. Therapeutic or pharmaceutical compositions may comprise oneor more lytic polypeptide(s), and optionally include natural, truncated,chimeric or shuffled lytic enzymes, optionally combined with othercomponents such as a carrier, vehicle, polypeptide, polynucleotide,holin protein(s), one or more antibiotics or suitable excipients,carriers or vehicles. The invention provides therapeutic compositions orpharmaceutical compositions of the lysins of the invention, includingPlySs2 and/or PlySs1 (particularly ΔPlySs1), for use in the killing,alleviation, decolonization, prophylaxis or treatment of gram-positivebacteria, including bacterial infections or related conditions. Theinvention provides therapeutic compositions or pharmaceuticalcompositions of the lysins of the invention, including PlySs2 and/orPlySs1 (particularly ΔPlySs1), for use in treating, reducing orcontrolling contamination and/or infections by gram positive bacteria,particularly including Streptococcus suis, including in contamination orinfection of or via an external surface such as skin. Compositions arethereby contemplated and provided for topical or dermatologicalapplications and general administration to the exterior, including theskin or other external surface. Compositions comprising PlySs2 or PlySs1lysin, including truncations or variants thereof, are provided hereinfor use in the killing, alleviation, decolonization, prophylaxis ortreatment of gram-positive bacteria, including bacterial infections orrelated conditions, particularly of Streptococcus, Staphylococcus,Enterococcus or Listeria, including Streptococcus pyogenes andantibiotic resistant Staphylococcus aureus.

The enzyme(s) or polypeptide(s) included in the therapeutic compositionsmay be one or more or any combination of unaltered phage associatedlytic enzyme(s), truncated lytic polypeptides, variant lyticpolypeptide(s), and chimeric and/or shuffled lytic enzymes.Additionally, different lytic polypeptide(s) genetically coded for bydifferent phage for treatment of the same bacteria may be used. Theselytic enzymes may also be any combination of “unaltered” lytic enzymesor polypeptides, truncated lytic polypeptide(s), variant lyticpolypeptide(s), and chimeric and shuffled lytic enzymes. The lyticenzyme(s)/polypeptide(s) in a therapeutic or pharmaceutical compositionfor gram-positive bacteria, including Streptococcus, Staphylococcus,Enterococcus and Listeria, may be used alone or in combination withantibiotics or, if there are other invasive bacterial organisms to betreated, in combination with other phage associated lytic enzymesspecific for other bacteria being targeted. The lytic enzyme, truncatedenzyme, variant enzyme, chimeric enzyme, and/or shuffled lytic enzymemay be used in conjunction with a holin protein. The amount of the holinprotein may also be varied. Various antibiotics may be optionallyincluded in the therapeutic composition with the enzyme(s) orpolypeptide(s) and with or without the presence of lysostaphin. Morethan one lytic enzyme or polypeptide may be included in the therapeuticcomposition.

The pharmaceutical composition can also include one or more alteredlytic enzymes, including isozymes, analogs, or variants thereof,produced by chemical synthesis or DNA recombinant techniques. Inparticular, altered lytic protein can be produced by amino acidsubstitution, deletion, truncation, chimerization, shuffling, orcombinations thereof. The pharmaceutical composition may contain acombination of one or more natural lytic protein and one or moretruncated, variant, chimeric or shuffled lytic protein. Thepharmaceutical composition may also contain a peptide or a peptidefragment of at least one lytic protein derived from the same ordifferent bacteria species, with an optional addition of one or morecomplementary agent, and a pharmaceutically acceptable carrier ordiluent.

The present invention provides to bacterial lysins comprising a PlySslysin polypeptide variant having bacterial killing activity. Theinvention describes PlySs lysin truncation mutants that contain only onecatalytic or enzymatic domain and retains gram positive antibacterialactivity. The invention describes, for example, exemplary PlySs lysintruncation mutant that contain only one domain selected from thepredicted alanine-amidase domain and the predicted glucosaminidasedomain. In the PLySS1 truncation mutant, for example, the C terminalglucosaminidase domain is deleted, so that the truncated lysin comprisesand contains an N-terminal enzymatic domain and a cell-wall bindingdomain. The ΔPlySS1 truncation has the N-terminal 254 amino acids,whereas the full length PlySs1 lysin has 452 amino acids. Thus, theinvention provides S suis lysin mutants, particularly PlySs1 lysinmutants which are truncated mutants containing only one catalytic domainand which retain killing activity against S. suis and numerous otherbacterial strains including other Streptococcus, as well asStaphylococcus, Listeria, and other bacteria, as provided anddemonstrated herein. A composition is herein provided comprising a PlySsmutant lysin, including a PlySS1 mutant lysin, having equal or greaterkilling activity against Streptococcus cells, including Streptococcussuis compared with the full length PlySs lysin protein, including thefull length PlySs1 lysin protein, the PlySs mutant lysin having apolypeptide variant of the amino acid sequence of SEQ ID NO:1 with amodification selected from the group consisting of: a) the PlySs mutantis a truncated mutant lysin containing only one catalytic domainselected from the group consisting of an endopeptidase domain and aglucosaminidase domain; b) the PlySs mutant is a truncated mutant lysinwithout a C-terminal enzymatic domain; c) the PlySs mutant has a singlecatalytic domain and a cell-wall binding domain; and d) the PlySs mutantcorresponds to SEQ ID NO:2, or amino acid variants thereof having one ormore conservative substitutions.

The therapeutic composition may also comprise a holin protein. Holinproteins (or “holins”) are proteins which produce holes in the cellmembrane. Holin proteins may form lethal membrane lesions that terminatecellular respiration in a bacteria. Like the lytic proteins, holinproteins are coded for and carried by a phage. In fact, it is quitecommon for the genetic code of the holin protein to be next to or evenwithin the code for the phage lytic protein. Most holin proteinsequences are short, and overall, hydrophobic in nature, with a highlyhydrophilic carboxy-terminal domain. In many cases, the putative holinprotein is encoded on a different reading frame within the enzymaticallyactive domain of the phage. In other cases, holin protein is encoded onthe DNA next or close to the DNA coding for the cell wall lytic protein.Holin proteins are frequently synthesized during the late stage of phageinfection and found in the cytoplasmic membrane where they causemembrane lesions. Holins can be grouped into two general classes basedon primary structure analysis. Class I holins are usually 95 residues orlonger and may have three potential transmembrane domains. Class IIholins are usually smaller, at approximately 65-95 residues, with thedistribution of charged and hydrophobic residues indicating two TMdomains (Young, et al. Trends in Microbiology v. 8, No. 4, March 2000).At least for the phages of gram-positive hosts, however, thedual-component lysis system may not be universal. Although the presenceof holins has been shown or suggested for several phages, no genes haveyet been found encoding putative holins for all phages. Holins have beenshown to be present in several bacteria, including, for example,lactococcal bacteriophage Tuc2009, lactococcal NLC3, pneumococcalbacteriophage EJ-1, Lactobacillus gasseri bacteriophage Nadh,Staphylococcus aureus bacteriophage Twort, Listeria monocytogenesbacteriophages, pneumococcal phage Cp-1, Bacillus subtillis phage M29,Lactobacillus delbrueckki bacteriophage LL-H lysin, and bacteriophage N11 of Staphyloccous aureus. (Loessner, et al., Journal of Bacteriology,August 1999, p. 4452-4460).

For example, holin proteins can be used in conjunction with the lyticenzymes to accelerate the speed and efficiency at which the bacteria arekilled. Holin proteins may also be in the form of chimeric and/orshuffled enzymes. Holin proteins may also be used alone in the treatmentof bacterial infections according to some embodiments.

The pharmaceutical composition can contain a complementary agent,including one or more antimicrobial agent and/or one or moreconventional antibiotics. In order to accelerate treatment of theinfection, the therapeutic agent may further include at least onecomplementary agent which can also potentiate the bactericidal activityof the lytic enzyme. Antimicrobials act largely by interfering with thestructure or function of a bacterial cell by inhibition of cell wallsynthesis, inhibition of cell-membrane function and/or inhibition ofmetabolic functions, including protein and DNA synthesis. Antibioticscan be subgrouped broadly into those affecting cell wall peptidoglycanbiosynthesis and those affecting DNA or protein synthesis in grampositive bacteria. Cell wall synthesis inhibitors, including penicillinand antibiotics like it, disrupt the rigid outer cell wall so that therelatively unsupported cell swells and eventually ruptures. Antibioticsaffecting cell wall peptidoglycan biosynthesis include: Glycopeptides,which inhibit peptidoglycan synthesis by preventing the incorporation ofN-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptidesubunits into the peptidoglycan matrix. Available glycopeptides includevancomycin and teicoplanin; Penicillins, which act by inhibiting theformation of peptidoglycan cross-links. The functional group ofpenicillins, the β-lactam moiety, binds and inhibits DD-transpeptidasethat links the peptidoglycan molecules in bacteria. Hydrolytic enzymescontinue to break down the cell wall, causing cytolysis or death due toosmotic pressure. Common penicillins include oxacillin, ampicillin andcloxacillin; and Polypeptides, which interfere with thedephosphorylation of the C₅₅-isoprenyl pyrophosphate, a molecule thatcarries peptidoglycan building-blocks outside of the plasma membrane. Acell wall-impacting polypeptide is bacitracin.

The complementary agent may be an antibiotic, such as erythromycin,clarithromycin, azithromycin, roxithromycin, other members of themacrolide family, penicilins, cephalosporins, and any combinationsthereof in amounts which are effective to synergistically enhance thetherapeutic effect of the lytic enzyme. Virtually any other antibioticmay be used with the altered and/or unaltered lytic enzyme. Similarly,other lytic enzymes may be included in the carrier to treat otherbacterial infections. Antibiotic supplements may be used in virtuallyall uses of the enzyme when treating different diseases. Thepharmaceutical composition can also contain a peptide or a peptidefragment of at least one lytic protein, one holin protein, or at leastone holin and one lytic protein, which lytic and holin proteins are eachderived from the same or different bacteria species, with an optionaladdition of a complementary agents, and a suitable carrier or diluent.

Also provided are compositions containing nucleic acid molecules that,either alone or in combination with other nucleic acid molecules, arecapable of expressing an effective amount of a lytic polypeptide(s) or apeptide fragment of a lytic polypeptide(s) in vivo. Cell culturescontaining these nucleic acid molecules, polynucleotides, and vectorscarrying and expressing these molecules in vitro or in vivo, are alsoprovided.

Therapeutic or pharmaceutical compositions may comprise lyticpolypeptide(s) combined with a variety of carriers to treat theillnesses caused by the susceptible gram-positive bacteria. The carriersuitably contains minor amounts of additives such as substances thatenhance isotonicity and chemical stability. Such materials are non-toxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, succinate, acetic acid, and otherorganic acids or their salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) polypeptides, e.g.,polyarginine or tripeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;glycine; amino acids such as glutamic acid, aspartic acid, histidine, orarginine; monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, glucose, mannose, trehalose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; counter-ions such as sodium; non-ionic surfactants such aspolysorbates, poloxamers, or polyethylene glycol (PEG); and/or neutralsalts, e.g., NaCl, KCl, MgCl.sub.2, CaCl.sub.2, and others. Glycerin orglycerol (1,2,3-propanetriol) is commercially available forpharmaceutical use. It may be diluted in sterile water for injection, orsodium chloride injection, or other pharmaceutically acceptable aqueousinjection fluid, and used in concentrations of 0.1 to 100% (v/v),preferably 1.0 to 50% more preferably about 20%. DMSO is an aproticsolvent with a remarkable ability to enhance penetration of many locallyapplied drugs. DMSO may be diluted in sterile water for injection, orsodium chloride injection, or other pharmaceutically acceptable aqueousinjection fluid, and used in concentrations of 0.1 to 100% (v/v). Thecarrier vehicle may also include Ringer's solution, a buffered solution,and dextrose solution, particularly when an intravenous solution isprepared.

Any of the carriers for the lytic polypeptide(s) may be manufactured byconventional means. However, it is preferred that any mouthwash orsimilar type products not contain alcohol to prevent denaturing of thepolyeptide/enzyme. Similarly, when the lytic polypeptide(s) is beingplaced in a cough drop, gum, candy or lozenge during the manufacturingprocess, such placement should be made prior to the hardening of thelozenge or candy but after the cough drop or candy has cooled somewhat,to avoid heat denaturation of the enzyme.

A lytic polypeptide(s) may be added to these substances in a liquid formor in a lyophilized state, whereupon it will be solubilized when itmeets body fluids such as saliva. The polypeptide(s)/enzyme may also bein a micelle or liposome.

The effective dosage rates or amounts of an altered or unaltered lyticenzyme/polypeptide(s) to treat the infection will depend in part onwhether the lytic enzyme/polypeptide(s) will be used therapeutically orprophylactically, the duration of exposure of the recipient to theinfectious bacteria, the size and weight of the individual, etc. Theduration for use of the composition containing the enzyme/polypeptide(s)also depends on whether the use is for prophylactic purposes, whereinthe use may be hourly, daily or weekly, for a short time period, orwhether the use will be for therapeutic purposes wherein a moreintensive regimen of the use of the composition may be needed, such thatusage may last for hours, days or weeks, and/or on a daily basis, or attimed intervals during the day. Any dosage form employed should providefor a minimum number of units for a minimum amount of time. Theconcentration of the active units of enzyme believed to provide for aneffective amount or dosage of enzyme may be in the range of about 100units/ml to about 500,000 units/ml of fluid in the wet or dampenvironment of the nasal and oral passages, and possibly in the range ofabout 100 units/ml to about 50,000 units/ml. More specifically, timeexposure to the active enzyme/polypeptide(s) units may influence thedesired concentration of active enzyme units per ml. Carriers that areclassified as “long” or “slow” release carriers (such as, for example,certain nasal sprays or lozenges) could possess or provide a lowerconcentration of active (enzyme) units per ml, but over a longer periodof time, whereas a “short” or “fast” release carrier (such as, forexample, a gargle) could possess or provide a high concentration ofactive (enzyme) units per ml, but over a shorter period of time. Theamount of active units per ml and the duration of time of exposuredepend on the nature of infection, whether treatment is to beprophylactic or therapeutic, and other variables. There are situationswhere it may be necessary to have a much higher unit/ml dosage or alower unit/ml dosage.

The lytic enzyme/polypeptide(s) should be in an environment having a pHwhich allows for activity of the lytic enzyme/polypeptide(s). Forexample if a human individual has been exposed to another human with abacterial upper respiratory disorder, the lytic enzyme/polypeptide(s)will reside in the mucosal lining and prevent any colonization of theinfecting bacteria. Prior to, or at the time the altered lytic enzyme isput in the carrier system or oral delivery mode, it is preferred thatthe enzyme be in a stabilizing buffer environment for maintaining a pHrange between about 4.0 and about 9.0, more preferably between about 5.5and about 7.5.

A stabilizing buffer may allow for the optimum activity of the lysinenzyme/polypeptide(s). The buffer may contain a reducing reagent, suchas dithiothreitol. The stabilizing buffer may also be or include a metalchelating reagent, such as ethylenediaminetetracetic acid disodium salt,or it may also contain a phosphate or citrate-phosphate buffer, or anyother buffer. The DNA coding of these phages and other phages may bealtered to allow a recombinant enzyme to attack one cell wall at morethan two locations, to allow the recombinant enzyme to cleave the cellwall of more than one species of bacteria, to allow the recombinantenzyme to attack other bacteria, or any combinations thereof. The typeand number of alterations to a recombinant bacteriophage produced enzymeare incalculable.

A mild surfactant can be included in a therapeutic or pharmaceuticalcomposition in an amount effective to potentiate the therapeutic effectof the lytic enzyme/polypeptide(s) may be used in a composition.Suitable mild surfactants include, inter alia, esters of polyoxyethylenesorbitan and fatty acids (Tween series), octylphenoxy polyethoxy ethanol(Triton-X series), n-Octyl-.beta.-D-glucopyranoside,n-Octyl-.beta.-D-thioglucopyranoside, n-Decyl-.beta.-D-glucopyranoside,n-Dodecyl-.beta.-D-glucopyranoside, and biologically occurringsurfactants, e.g., fatty acids, glycerides, monoglycerides, deoxycholateand esters of deoxycholate.

Preservatives may also be used in this invention and preferably compriseabout 0.05% to 0.5% by weight of the total composition. The use ofpreservatives assures that if the product is microbially contaminated,the formulation will prevent or diminish microorganism growth. Somepreservatives useful in this invention include methylparaben,propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDMHydantoin, 3-Iodo-2-Propylbutyl carbamate, potassium sorbate,chlorhexidine digluconate, or a combination thereof.

Pharmaceuticals for use in all embodiments of the invention includeantimicrobial agents, anti-inflammatory agents, antiviral agents, localanesthetic agents, corticosteroids, destructive therapy agents,antifungals, and antiandrogens. In the treatment of acne, activepharmaceuticals that may be used include antimicrobial agents,especially those having anti-inflammatory properties such as dapsone,erythromycin, minocycline, tetracycline, clindamycin, and otherantimicrobials. The preferred weight percentages for the antimicrobialsare 0.5% to 10%.

Local anesthetics include tetracaine, tetracaine hydrochloride,lidocaine, lidocaine hydrochloride, dyclonine, dyclonine hydrochloride,dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride,butambenpicrate, and pramoxine hydrochloride. A preferred concentrationfor local anesthetics is about 0.025% to 5% by weight of the totalcomposition. Anesthetics such as benzocaine may also be used at apreferred concentration of about 2% to 25% by weight.

Corticosteroids that may be used include betamethasone dipropionate,fluocinolone actinide, betamethasone valerate, triamcinolone actinide,clobetasol propionate, desoximetasone, diflorasone diacetate,amcinonide, flurandrenolide, hydrocortisone valerate, hydrocortisonebutyrate, and desonide are recommended at concentrations of about 0.01%to 1.0% by weight. Preferred concentrations for corticosteroids such ashydrocortisone or methylprednisolone acetate are from about 0.2% toabout 5.0% by weight.

Additionally, the therapeutic composition may further comprise otherenzymes, such as the enzyme lysostaphin for the treatment of anyStaphylococcus aureus bacteria present along with the susceptiblegram-positive bacteria. Mucolytic peptides, such as lysostaphin, havebeen suggested to be efficacious in the treatment of S. aureusinfections of humans (Schaffner et al., Yale J. Biol. & Med., 39:230(1967). Lysostaphin, a gene product of Staphylococcus simulans, exerts abacteriostatic and bactericidal effect upon S. aureus by enzymaticallydegrading the polyglycine crosslinks of the cell wall (Browder et al.,Res. Comm., 19: 393-400 (1965)). U.S. Pat. No. 3,278,378 describesfermentation methods for producing lysostaphin from culture media of S.staphylolyticus, later renamed S. simulans. Other methods for producinglysostaphin are further described in U.S. Pat. Nos. 3,398,056 and3,594,284. The gene for lysostaphin has subsequently been cloned andsequenced (Recsei et al., Proc. Natl. Acad. Sci. USA, 84: 1127-1131(1987)). The recombinant mucolytic bactericidal protein, such asr-lysostaphin, can potentially circumvent problems associated withcurrent antibiotic therapy because of its targeted specificity, lowtoxicity and possible reduction of biologically active residues.Furthermore, lysostaphin is also active against non-dividing cells,while most antibiotics require actively dividing cells to mediate theireffects (Dixon et al., Yale J. Biology and Medicine, 41: 62-68 (1968)).Lysostaphin, in combination with the altered lytic enzyme, can be usedin the presence or absence of antibiotics. There is a degree of addedimportance in using both lysostaphin and the lysin enzyme in the sametherapeutic agent. Frequently, when a human has a bacterial infection,the infection by one genus of bacteria weakens the human body or changesthe bacterial flora of the body, allowing other potentially pathogenicbacteria to infect the body. One of the bacteria that sometimesco-infects a body is Staphylococcus aureus. Many strains ofStaphylococcus aureus produce penicillinase, such that Staphylococcus,Streptococcus, and other Gram positive bacterial strains will not bekilled by standard antibiotics. Consequently, the use of the lysin andlysostaphin, possibly in combination with antibiotics, can serve as themost rapid and effective treatment of bacterial infections. Atherapeutic composition may also include mutanolysin, and lysozyme.

Means of application of the therapeutic composition comprising a lyticenzyme/polypeptide(s) include, but are not limited to direct, indirect,carrier and special means or any combination of means. Directapplication of the lytic enzyme/polypeptide(s) may be by any suitablemeans to directly bring the polypeptide in contact with the site ofinfection or bacterial colonization, such as to the nasal area (forexample nasal sprays), dermal or skin applications (for example topicalointments or formulations), suppositories, tampon applications, etc.Nasal applications include for instance nasal sprays, nasal drops, nasalointments, nasal washes, nasal injections, nasal packings, bronchialsprays and inhalers, or indirectly through use of throat lozenges,mouthwashes or gargles, or through the use of ointments applied to thenasal nares, or the face or any combination of these and similar methodsof application. The forms in which the lytic enzyme may be administeredinclude but are not limited to lozenges, troches, candies, injectants,chewing gums, tablets, powders, sprays, liquids, ointments, andaerosols.

When the natural and/or altered lytic enzyme(s)/polypeptide(s) isintroduced directly by use of sprays, drops, ointments, washes,injections, packing and inhalers, the enzyme is preferably in a liquidor gel environment, with the liquid acting as the carrier. A dryanhydrous version of the altered enzyme may be administered by theinhaler and bronchial spray, although a liquid form of delivery ispreferred.

Compositions for treating topical infections or contaminations comprisean effective amount of at least one lytic enzyme, including PlySs1and/or PlySs2, according to the invention and a carrier for deliveringat least one lytic enzyme to the infected or contaminated skin, coat, orexternal surface of a companion animal or livestock. The mode ofapplication for the lytic enzyme includes a number of different typesand combinations of carriers which include, but are not limited to anaqueous liquid, an alcohol base liquid, a water soluble gel, a lotion,an ointment, a nonaqueous liquid base, a mineral oil base, a blend ofmineral oil and petrolatum, lanolin, liposomes, protein carriers such asserum albumin or gelatin, powdered cellulose carmel, and combinationsthereof. A mode of delivery of the carrier containing the therapeuticagent includes, but is not limited to a smear, spray, a time-releasepatch, a liquid absorbed wipe, and combinations thereof. The lyticenzyme may be applied to a bandage either directly or in one of theother carriers. The bandages may be sold damp or dry, wherein the enzymeis in a lyophilized form on the bandage. This method of application ismost effective for the treatment of infected skin. The carriers oftopical compositions may comprise semi-solid and gel-like vehicles thatinclude a polymer thickener, water, preservatives, active surfactants oremulsifiers, antioxidants, sun screens, and a solvent or mixed solventsystem. U.S. Pat. No. 5,863,560 (Osborne) discusses a number ofdifferent carrier combinations which can aid in the exposure of the skinto a medicament. Polymer thickeners that may be used include those knownto one skilled in the art, such as hydrophilic and hydroalcoholicgelling agents frequently used in the cosmetic and pharmaceuticalindustries. CARBOPOL® is one of numerous cross-linked acrylic acidpolymers that are given the general adopted name carbomer. Thesepolymers dissolve in water and form a clear or slightly hazy gel uponneutralization with a caustic material such as sodium hydroxide,potassium hydroxide, triethanolamine, or other amine bases. KLUCEL® T isa cellulose polymer that is dispersed in water and forms a uniform gelupon complete hydration. Other preferred gelling polymers includehydroxyethylcellulose, cellulose gum, MVE/MA decadiene crosspolymer,PVM/MA copolymer, or a combination thereof.

A composition comprising a lytic enzyme/polypeptide(s) can beadministered in the form of a candy, chewing gum, lozenge, troche,tablet, a powder, an aerosol, a liquid, a liquid spray, or toothpastefor the prevention or treatment of bacterial infections associated withupper respiratory tract illnesses. The lozenge, tablet, or gum intowhich the lytic enzyme/polypeptide(s) is added may contain sugar, cornsyrup, a variety of dyes, non-sugar sweeteners, flavorings, any binders,or combinations thereof. Similarly, any gum-based products may containacacia, carnauba wax, citric acid, cornstarch, food colorings,flavorings, non-sugar sweeteners, gelatin, glucose, glycerin, gum base,shellac, sodium saccharin, sugar, water, white wax, cellulose, otherbinders, and combinations thereof. Lozenges may further contain sucrose,cornstarch, acacia, gum tragacanth, anethole, linseed, oleoresin,mineral oil, and cellulose, other binders, and combinations thereof.Sugar substitutes can also be used in place of dextrose, sucrose, orother sugars.

Compositions comprising lytic enzymes, or their peptide fragments can bedirected to the mucosal lining, where, in residence, they killcolonizing disease bacteria. The mucosal lining, as disclosed anddescribed herein, includes, for example, the upper and lower respiratorytract, eye, buccal cavity, nose, rectum, vagina, periodontal pocket,intestines and colon. Due to natural eliminating or cleansing mechanismsof mucosal tissues, conventional dosage forms are not retained at theapplication site for any significant length of time.

It may be advantageous to have materials which exhibit adhesion tomucosal tissues, to be administered with one or more phage enzymes andother complementary agents over a period of time. Materials havingcontrolled release capability are particularly desirable, and the use ofsustained release mucoadhesives has received a significant degree ofattention. J. R. Robinson (U.S. Pat. No. 4,615,697, incorporated hereinby reference) provides a good review of the various controlled releasepolymeric compositions used in mucosal drug delivery. The patentdescribes a controlled release treatment composition which includes abioadhesive and an effective amount of a treating agent. The bioadhesiveis a water swellable, but water insoluble fibrous, crosslinked, carboxyfunctional polymer containing (a) a plurality of repeating units ofwhich at least about 80 percent contain at least one carboxylfunctionality, and (b) about 0.05 to about 1.5 percent crosslinkingagent substantially free from polyalkenyl polyether. While the polymersof Robinson are water swellable but insoluble, they are crosslinked, notthermoplastic, and are not as easy to formulate with active agents, andinto the various dosage forms, as the copolymer systems of the presentapplication. Micelles and multilamillar micelles may also be used tocontrol the release of enzyme.

Other approaches involving mucoadhesives which are the combination ofhydrophilic and hydrophobic materials, are known. Orahesive® from E.R.Squibb & Co is an adhesive which is a combination of pectin, gelatin,and sodium carboxymethyl cellulose in a tacky hydrocarbon polymer, foradhering to the oral mucosa. However, such physical mixtures ofhydrophilic and hydrophobic components eventually fall apart. Incontrast, the hydrophilic and hydrophobic domains in this applicationproduce an insoluble copolymer. U.S. Pat. No. 4,948,580, alsoincorporated by reference, describes a bioadhesive oral drug deliverysystem. The composition includes a freeze-dried polymer mixture formedof the copolymer poly(methyl vinyl ether/maleic anhydride) and gelatin,dispersed in an ointment base, such as mineral oil containing dispersedpolyethylene. U.S. Pat. No. 5,413,792 (incorporated herein by reference)discloses paste-like preparations comprising (A) a paste-like basecomprising a polyorganosiloxane and a water soluble polymeric materialwhich are preferably present in a ratio by weight from 3:6 to 6:3, and(B) an active ingredient. U.S. Pat. No. 5,554,380 claims a solid orsemisolid bioadherent orally ingestible drug delivery system containinga water-in-oil system having at least two phases. One phase comprisesfrom about 25% to about 75% by volume of an internal hydrophilic phaseand the other phase comprises from about 23% to about 75% by volume ofan external hydrophobic phase, wherein the external hydrophobic phase iscomprised of three components: (a) an emulsifier, (b) a glyceride ester,and (c) a wax material. U.S. Pat. No. 5,942,243 describes somerepresentative release materials useful for administering antibacterialagents, which are incorporated by reference.

Therapeutic or pharmaceutical compositions can also contain polymericmucoadhesives including a graft copolymer comprising a hydrophilic mainchain and hydrophobic graft chains for controlled release ofbiologically active agents. The graft copolymer is a reaction product of(1) a polystyrene macromonomer having an ethylenically unsaturatedfunctional group, and (2) at least one hydrophilic acidic monomer havingan ethylenically unsaturated functional group. The graft chains consistessentially of polystyrene, and the main polymer chain of hydrophilicmonomeric moieties, some of which have acidic functionality. The weightpercent of the polystyrene macromonomer in the graft copolymer isbetween about 1 and about 20% and the weight percent of the totalhydrophilic monomer in the graft copolymer is between 80 and 99%, andwherein at least 10% of said total hydrophilic monomer is acidic, saidgraft copolymer when fully hydrated having an equilibrium water contentof at least 90%. Compositions containing the copolymers graduallyhydrate by sorption of tissue fluids at the application site to yield avery soft jelly like mass exhibiting adhesion to the mucosal surface.During the period of time the composition is adhering to the mucosalsurface, it provides sustained release of the pharmacologically activeagent, which is absorbed by the mucosal tissue.

The compositions of this application may optionally contain otherpolymeric materials, such as poly(acrylic acid), poly, -(vinylpyrrolidone), and sodium carboxymethyl cellulose plasticizers, and otherpharmaceutically acceptable excipients in amounts that do not causedeleterious effect upon mucoadhesivity of the composition.

The dosage forms of the compositions of this invention can be preparedby conventional methods. In cases where intramuscular injection is thechosen mode of administration, an isotonic formulation is preferablyused. Generally, additives for isotonicity can include sodium chloride,dextrose, mannitol, sorbitol and lactose. In some cases, isotonicsolutions such as phosphate buffered saline are preferred. Stabilizersinclude gelatin and albumin. A vasoconstriction agent can be added tothe formulation. The pharmaceutical preparations according to thisapplication are provided sterile and pyrogen free.

A lytic enzyme/polypeptide(s) of the invention may also be administeredby any pharmaceutically applicable or acceptable means includingtopically, orally or parenterally. For example, the lyticenzyme/polypeptide(s) can be administered intramuscularly,intrathecally, subdermally, subcutaneously, or intravenously to treatinfections by gram-positive bacteria. In cases where parenteralinjection is the chosen mode of administration, an isotonic formulationis preferably used. Generally, additives for isotonicity can includesodium chloride, dextrose, mannitol, sorbitol and lactose. In somecases, isotonic solutions such as phosphate buffered saline arepreferred. Stabilizers include gelatin and albumin. A vasoconstrictionagent can be added to the formulation. The pharmaceutical preparationsaccording to this application are provided sterile and pyrogen free.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The effective dosage rates or amounts of the lytic enzyme/polypeptide(s)to be administered parenterally, and the duration of treatment willdepend in part on the seriousness of the infection, the weight of thepatient, particularly human, the duration of exposure of the recipientto the infectious bacteria, the number of square centimeters of skin ortissue which are infected, the depth of the infection, the seriousnessof the infection, and a variety of a number of other variables. Thecomposition may be applied anywhere from once to several times a day,and may be applied for a short or long term period. The usage may lastfor days or weeks. Any dosage form employed should provide for a minimumnumber of units for a minimum amount of time. The concentration of theactive units of enzymes believed to provide for an effective amount ordosage of enzymes may be selected as appropriate. The amount of activeunits per ml and the duration of time of exposure depend on the natureof infection, and the amount of contact the carrier allows the lyticenzyme(s)/polypeptide(s) to have.

Methods and Assays

The bacterial killing capability, and indeed the significantly broadrange of bacterial killing, exhibited by the lysin polypeptide(s) of theinvention provides for various methods based on the antibacterialeffectiveness of the polypeptide(s) of the invention. Thus, the presentinvention contemplates antibacterial methods, including methods forkilling of gram-positive bacteria, for reducing a population ofgram-positive bacteria, for treating or alleviating a bacterialinfection, for treating a human subject exposed to a pathogenicbacteria, and for treating a human subject at risk for such exposure.The susceptible bacteria are demonstrated herein to include the bacteriafrom which the phage enzyme(s) of the invention are originally derived,Streptococcus suis, as well as various other Streptococcal,Staphylococcal, Enterococcal and Listeria bacterial strains. Methods oftreating various conditions are also provided, including methods ofprophylactic treatment of Streptococcal, Staphylococcal, Enterococcal orListeria infections, treatment of Streptococcal, Staphylococcal,Enterococcal or Listeria infections, reducing Streptococcal,Staphylococcal, Enterococcal or Listeria population or carriage,treating lower respiratory infection, treating ear infection, treatingottis media, treating endocarditis, and treating or preventing otherlocal or systemic infections or conditions.

The lysin(s) of the present invention demonstrate remarkable capabilityto kill and effectiveness against bacteria from various species such asmultiple Streptococcal or Staphylococcal species, bacteria acrossdistinct species groups such as bacteria from each of Streptococcal,Staphylococcal, Enterococcal and/or Listeria, and bacterial fromdistinct orders. The bacterial taxonomic class of Bacilli includes twoorders, Bacillales and Lactobacillales. The Bacillales order includesStaphylococcus, Listeria and also Bacillus. The Lactobacillales orderincludes Streptococcus, Enterococcus, Lactobacillus and Lactococcus. Thelysin(s) of the present invention demonstrate anti-bacterial activityand the capability to kill bacteria from distinct orders of bacteria,particularly from distinct orders of Bacilli bacteria. The lysin(s)provided herein are capable of killing bacteria from order Bacillalesand also from order Lactobacillales. The PlySs2 lysin is demonstratedherein to kill bacteria from two distinct orders, particularlyBacillales and Lactobacillales, in vitro and in vivo. Lysin of thepresent invention is capable of killing Bacillales and Lactobacillalesbacteria in mixed culture and in mixed infections in vivo. The inventionthus contemplates treatment, decolonization, and/or decontamination ofbacteria, cultures or infections or in instances wherein more than onegram positive bacteria is suspected or present. In particular, theinvention contemplates treatment, decolonization, and/or decontaminationof bacteria, cultures or infections or in instances wherein more thanone type of Bacilalles bacteria, more than one type of Lactobacillalesbacteria, or at least one type of Bacillales and one type ofLactobacillales bacteria is suspected, present, or may be present.

This invention may also be used to treat septicemia, particularly in ahuman. For the treatment of a septicemic infection, such as forpneumoniae, or bacterial meningitis, there should be a continuousintravenous flow of therapeutic agent into the blood stream. Theconcentration of the enzymes for the treatment of septicemia isdependent upon the bacterial count in the blood and the blood volume.

Also provided is a method for treating Streptococcal, Staphylococcal,Enterococcal or Listeria infection, carriage or populations comprisestreating the infection with a therapeutic agent comprising an effectiveamount of at least one lytic enzyme(s)/polypeptide(s) of the invention,particularly PlySs2 and/or PlySs1, particularly PlySs2. Morespecifically, lytic enzyme/polypeptide capable of lysing the cell wallof Streptococcal, Staphylococcal, Enterococcal or Listeria bacterialstrains is produced from genetic material from a bacteriophage specificfor Streptococcus suis. In the methods of the invention, the lysinpolypeptide(s) of the present invention, including PlySs2 and/or PlySs1,particularly PlySs2, are useful and capable in prophylactic andtreatment methods directed against gram-positive bacteria, particularlyStreptococcal, Staphylococcal, Enterococcal or Listeria infections orbacterial colonization. Bacterial strains susceptible and relevant astargets in the methods of the invention include and may be selected fromStaphylococcus aureus, Listeria monocytogenes, Staphylococcus simulans,Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi,Streptococcus equi zoo, Streptococcus agalactiae (GBS), Streptococcuspyogenes (GAS), Streptococcus sanguinis, Streptococcus gordonii,Streptococcus dysgalactiae, Group G Streptococcus, Group EStreptococcus, Enterococcus faecalis and Streptococcus pneumonia.

The invention includes methods of treating or alleviating Streptococcal,including S. pyogenes, and/or Staphylococcal, including S. aureus,related infections or conditions, including antibiotic-resistantStaphylococcus aureus, particularly including MRSA, wherein the bacteriaor a human subject infected by or exposed to the particular bacteria, orsuspected of being exposed or at risk, is contacted with or administeredan amount of isolated lysin polypeptide(s) of the invention effective tokill the particular bacteria. Thus, one or more of PlySs2 and/or PlySs1,including truncations or variants thereof, including such polypeptidesas provided herein in FIGS. 3 and 4 and in SEQ ID NOS: 1, 2 or 3, iscontacted or administered so as to be effective to kill the relevantbacteria or otherwise alleviate or treat the bacterial infection.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds, addedadditional compound(s), or lysin enzyme compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

The term ‘assay’ means any process used to measure a specific propertyof a compound. A ‘screening assay’ means a process used to characterizeor select compounds based upon their activity from a collection ofcompounds.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to and encompassed in the term‘prevention’, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

‘Therapeutically effective amount’ means that amount of a drug,compound, antimicrobial, antibody, polypeptide, or pharmaceutical agentthat will elicit the biological or medical response of a subject that isbeing sought by a medical doctor or other clinician. In particular, withregard to gram-positive bacterial infections and growth of gram-positivebacteria, the term “effective amount” is intended to include aneffective amount of a compound or agent that will bring about abiologically meaningful decrease in the amount of or extent of infectionof gram-positive bacteria, including having a bacteriocidal and/orbacteriostatic effect. The phrase “therapeutically effective amount” isused herein to mean an amount sufficient to prevent, and preferablyreduce by at least about 30 percent, more preferably by at least 50percent, most preferably by at least 90 percent, a clinicallysignificant change in the growth or amount of infectious bacteria, orother feature of pathology such as for example, elevated fever or whitecell count as may attend its presence and activity.

The term ‘treating’ or ‘treatment’ of any disease or infection refers,in one embodiment, to ameliorating the disease or infection (i.e.,arresting the disease or growth of the infectious agent or bacteria orreducing the manifestation, extent or severity of at least one of theclinical symptoms thereof). In another embodiment ‘treating’ or‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or infection,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In a further embodiment, ‘treating’ or ‘treatment’ relates to slowingthe progression of a disease or reducing an infection.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

It is noted that in the context of treatment methods which are carriedout in vivo or medical and clinical treatment methods in accordance withthe present application and claims, the term subject, patient orindividual is intended to refer to a human.

The terms “gram-positive bacteria”, “Gram-positive bacteria”,“gram-positive” and any variants not specifically listed, may be usedherein interchangeably, and as used throughout the present applicationand claims refer to Gram-positive bacteria which are known and/or can beidentified by the presence of certain cell wall and/or cell membranecharacteristics and/or by staining with Gram stain. Gram positivebacteria are known and can readily be identified and may be selectedfrom but are not limited to the genera Listeria, Staphylococcus,Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, andClostridium, and include any and all recognized or unrecognized speciesor strains thereof. In an aspect of the invention, the PlyS lysinsensitive gram-positive bacteria include bacteria selected from one ormore of Listeria, Staphylococcus, Streptococcus, and Enterococcus.

The term “bacteriocidal” refers to capable of killing bacterial cells.

The term “bacteriostatic” refers to capable of inhibiting bacterialgrowth, including inhibiting growing bacterial cells.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in the S phaseactivity of a target cellular mass, or other feature of pathology suchas for example, elevated blood pressure, fever or white cell count asmay attend its presence and activity.

One method for treating systemic or tissue bacterial infections causedby Streptococcus or Staphylococcus bacteria comprises parenterallytreating the infection with a therapeutic agent comprising an effectiveamount of one or more lysin polypeptide(s) of the invention,particularly PlySs2 and/or PlySs1, including truncations or variantsthereof, including such polypeptides as provided herein in FIGS. 3 and 4and in SEQ ID NOS: 1, 2 or 3 and an appropriate carrier. A number ofother different methods may be used to introduce the lyticenzyme(s)/polypeptide(s). These methods include introducing the lyticenzyme(s)/polypeptide(s) intravenously, intramuscularly, subcutaneously,intrathecally, and subdermally. One skilled in the art, includingmedical personnel, will be capable of evaluating and recognizing themost appropriate mode or means of administration, given the nature andextent of the bacterial condition and the strain or type of bacteriainvolved or suspected. For instance, intrathecal use and administrationof one or more lytic polypeptide(s) would be most beneficial fortreatment of bacterial meningitis.

Infections may be also be treated by injecting into the infected tissueof the human patient a therapeutic agent comprising the appropriatelytic enzyme(s)/polypeptide(s) and a carrier for the enzyme. The carriermay be comprised of distilled water, a saline solution, albumin, aserum, or any combinations thereof. More specifically, solutions forinfusion or injection may be prepared in a conventional manner, e.g.with the addition of preservatives such as p-hydroxybenzoates orstabilizers such as alkali metal salts of ethylene-diamine tetraaceticacid, which may then be transferred into fusion vessels, injection vialsor ampules. Alternatively, the compound for injection may be lyophilizedeither with or without the other ingredients and be solubilized in abuffered solution or distilled water, as appropriate, at the time ofuse. Non-aqueous vehicles such as fixed oils, liposomes, and ethyloleate are also useful herein. Other phage associated lytic enzymes,along with a holin protein, may be included in the composition.

Various methods of treatment are provided for using a lyticenzyme/polypeptide(s), such as PlySs2 and PlySS1 as exemplified herein,as a prophylactic treatment for eliminating or reducing the carriage ofsusceptible bacteria, preventing those humans who have been exposed toothers who have the symptoms of an infection from getting sick, or as atherapeutic treatment for those who have already become ill from theinfection. Similarly, the lytic enzyme(s)/polypeptide(s) can be used totreat, for example, lower respiratory tract illnesses, particularly bythe use of bronchial sprays or intravenous administration of the enzyme.For example, a lytic enzyme can be used for the prophylactic andtherapeutic treatment of eye infections, such as conjunctivitis. Themethod of treatment comprises administering eye drops or an eye washwhich comprise an effective amount of at least one lytic polypeptide(s)of the invention and a carrier capable of being safely applied to aneye, with the carrier containing the lytic enzymes. The eye drops or eyewash are preferably in the form of an isotonic solution. The pH of thesolution should be adjusted so that there is no irritation of the eye,which in turn would lead to possible infection by other organisms, andpossible to damage to the eye. While the pH range should be in the samerange as for other lytic enzymes, the most optimal pH will be in therange as demonstrated and provided herein. Similarly, buffers of thesort described above for the other lytic enzymes should also be used.Other antibiotics which are suitable for use in eye drops may be addedto the composition containing the enzymes. Bactericides andbacteriostatic compounds may also be added. The concentration of theenzyme(s) in the solution can be in the range of from about 100 units/mlto about 500,000 units/ml, with a more preferred range of about 100 toabout 5,000 units/mil, and about 100 to about 50,000 units/ml.Concentrations can be higher or lower than the ranges provided.

The lytic polypeptide(s) of the invention may also be used in a contactlens solution, for the soaking and cleaning of contact lenses. Thissolution, which is normally an isotonic solution, may contain, inaddition to the enzyme, sodium chloride, mannitol and other sugaralcohols, borates, preservatives, and the like. A lyticenzyme/polypeptide of the invention may also be administered to the earof a patient. Thus, for instance a lytic polypeptide(s) of the inventionmay be used to treat ear infections, for example caused by Streptococcuspneumoniae. Otitis media is an inflammation of the middle earcharacterized by symptoms such as otalgia, hearing loss and fever. Oneof the primary causes of these symptoms is a build up of fluid(effusion) in the middle ear. Complications include permanent hearingloss, perforation of the tympanic membrane, acquired cholesteatoma,mastoiditis, and adhesive otitis. Children who develop otitis media inthe first years of life are at risk for recurrent acute or chronicdisease. One of the primary causes of otitis media is Streptococcuspneumoniae. The lytic enzyme(s)/polypeptide(s) may be applied to aninfected ear by delivering the enzyme(s) in an appropriate carrier tothe canal of the ear. The carrier may comprise sterile aqueous or oilysolutions or suspensions. The lytic enzyme(s) may be added to thecarrier, which may also contain suitable preservatives, and preferably asurface-active agent. Bactericidal and fungicidal agents preferablyincluded in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol. Additionally, any numberof other eardrop carriers may be used. The concentrations andpreservatives used for the treatment of otitis media and other similarear infections are the same as discussed for eye infections, and thecarrier into which the enzyme goes is similar or identical to thecarriers for treatment of eye infections. Additionally, the carrier maytypically includes vitamins, minerals, carbohydrates, sugars, aminoacids, proteinaceous materials, fatty acids, phospholipids,antioxidants, phenolic compounds, isotonic solutions, oil basedsolutions, oil based suspensions, and combinations thereof.

The diagnostic, prophylactic and therapeutic possibilities andapplications that are raised by the recognition of and isolation of thelysin polypeptide(s) of the invention, derive from the fact that thepolypeptides of the invention cause direct and specific effects (e.g.killing) in susceptible bacteria. Thus, the polypeptides of theinvention may be used to eliminate, characterize, or identify therelevant and susceptible bacteria.

Thus, a diagnostic method of the present invention may compriseexamining a cellular sample or medium for the purpose of determiningwhether it contains susceptible bacteria, or whether the bacteria in thesample or medium are susceptible by means of an assay including aneffective amount of one or more lysin polypeptide(s) and a means forcharacterizing one or more cell in the sample, or for determiningwhether or not cell lysis has occurred or is occurring. Patients capableof benefiting from this method include those suffering from anundetermined infection, a recognized bacterial infection, or suspectedof being exposed to or carrying a particular bacteria. A fluid, food,medical device, composition or other such sample which will come incontact with a subject or patient may be examined for susceptiblebacteria or may be eliminated of relevant bacteria. In one such aspect afluid, food, medical device, composition or other such sample may besterilized or otherwise treated to eliminate or remove any potentialrelevant bacteria by incubation with or exposure to one or more lyticpolypeptide(s) of the invention.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. In one instance, the lytic polypeptide(s) of theinvention complex(es) with or otherwise binds or associates withrelevant or susceptible bacteria in a sample and one member of thecomplex is labeled with a detectable label. The fact that a complex hasformed and, if desired, the amount thereof, can be determined by knownmethods applicable to the detection of labels. The labels most commonlyemployed for these studies are radioactive elements, enzymes, chemicalswhich fluoresce when exposed to ultraviolet light, and others. A numberof fluorescent materials are known and can be utilized as labels. Theseinclude, for example, fluorescein, rhodamine, auramine, Texas Red, AMCAblue and Lucifer Yellow. The radioactive label can be detected by any ofthe currently available counting procedures. The preferred isotope maybe selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y,¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Enzyme labels are likewise useful, and can bedetected by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques. The enzyme is conjugated to the selected particle byreaction with bridging molecules such as carbodiimides, diisocyanates,glutaraldehyde and the like. Many enzymes which can be used in theseprocedures are known and can be utilized. The preferred are peroxidase,ß-glucuronidase, ß-D-glucosidase, ß-D-galactosidase, urease, glucoseoxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos.3,654,090; 3,850,752; and 4,016,043 are referred to by way of examplefor their disclosure of alternate labeling material and methods.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1 Cloning and Characterization of Phage Lysins from S. suis

Streptococcus suis is a Gram-positive pathogen that infects pigsworldwide. Reports of zoonotic transmission from pigs to humans areincreasing (Sriskandan S. et al (2006) PLoS Medicine 3(5):585-567). S.suis may develop a consistent presence in human populations in years tocome. Humans and pigs have been treated with penicillin or gentamicin,but S. suis isolates resistant to these antibiotics exist (Cantin, M. etal (1992) J Vet Diagnostic Investig 4:170-174).

We purified and characterized two phage lysins from strains of S. suis(PlySs1 and PlySs2) and confirmed their in vitro activity againstvarious S. suis strains. In addition, the S. suis lysin, particularlyPlySs2 lysin, was shown in vitro to kill various other and distinctstrains of Streptococcus, including Group B strep. The PlySs2 lysin alsois effective in killing numerous other bacteria, including otherpathogenic and clinically significant bacteria, particularlyStaphylococcus, including Staphylococccus aureus, even antibioticresistant S. aureus such as MRSA, Enterococcus, including Enterococcusfaecalis, and Listeria.

Results

PlySs1 was isolated and cloned via a functional genomic screen using S.suis prophage genomic DNA and PlySs2 was identified by sequence analysisof the S. suis prophage genome sequence and then isolated and cloned.The PlySs1 lysin was cloned through functional shotgun screening of thegenome of S. suis 7711, a serotype 7 strain. Microgram quantities ofgenomic DNA (gDNA) were briefly subjected to restriction digestion withTsp509I (NEB). Fragments 1.5-4 kb in length were isolated viaagarose-gel electrophoresis and ligated into EcoRI-linearized pBAD24plasmid. This plasmid confers ampicillin resistance and allows forarabinose induction of the recombinant insert. To identifylysin-encoding clones, libraries were subject to a novel screeningtechnique that relies upon the toxicity of adjacently-encoded holinproteins (Schmitz J. E. et al (2010) Adv Environ Microbiol76(21):7181-7187). Briefly, E. coli TOP10 transformants were plated ontoLB-agar supplemented with ampicillin and sheep's blood. Followingproliferation to macroscopic colonies, the plates were exposed to a mistof arabinose to induce recombinant transcription. Toxic clones wererevealed by the development of a surrounding zone of hemolysis. Thesecolonies were identified, re-propagated and subject to a secondaryscreen in which they were overlaid with heat-killed bacteria (to assaydirectly for the production of lytic enzyme). For the S. suis strain(7711) that yielded the PlySs1 lysin, ˜3,500 clones were subjected tothe original hemolysis screen; 100 of these were selected for thesecondary screen, 2 of which encoded the lytic enzyme. For thetheoretical translated protein, putative enzymatic and binding domainsassignments were made via Pfam analysis (pfam.sanger.ac.uk). Based onthis information, primers were designed for synthesizing a truncatedconstructed (hereafter referred to as PlySs1) with an inserted stopcodon preceding the C-terminal glucosaminidase domain. The nucleic acidand amino acid sequences of the full length PlySs1 lysin and the aminoacid sequence of a truncated enzyme are provided in FIG. 3.

For the identification and cloning of PlySs2, the genomes of 8 sequencedisolates of S. suis were inspected for the presence of lysin-encodinggenes within integrated prophage. These strains were: 05ZYH33 (NCBIGenome Project #17153); 98HAH33 (#17155); BM407 (#32237); GZ1 (#18737);P1/7 (#352); SC84 (#32239); 05HAS68 05HAH33 (#17157); and 89/1591(#12417). For each genome, the topologically-arranged list of annotatedORFs was manually inspected for potential prophage regions. If aprophage was suspected, the theoretical translations of each ORF in thatregion were subject to, and putative lysin-status was assigned based onthe combination of predicted enzymatic and binding domains. The onlylysin gene identified in this manner (PlySs2 from strain 89/1591) wasPCR-cloned from genomic DNA and cloned in to the pBad24 E. coliexpression plasmid (see below). The nucleotide and amino acid sequenceof PlySs2 lysin are provided in FIG. 4.

As described above, two S. suis lysins have been identified and clonedthrough a combination of functional recombinant screening andcomputational analysis of published S. suis genomes. These lysins havebeen cloned and named PlySs1 and PlySs2. Like other lysins, the S. suislysins, particularly PlySs2, have an N-terminal catalytic domain andC-terminal cell-binding domain (SH-3 Type 5 binding domain in PlySs2)(FIG. 2). In fact, the natural structure of PlySs1 as cloned from S.suis strain 7711 contained an additional secondary catalytic domaindownstream of the binding domain (an atypical lysin arrangement),however this domain was recombinantly eliminated (as described above) toconform to standard architecture.

The lysin-encoding gene PlySs2 was found within an integrated prophagegenome along the sequenced genome of S. suis serotype 2 strain 89/1591(NCBI Genome Project #12417, GenBank accession ZP_03625529) (Lucas, S.et al, US DOE Joint Genome Institute, direct submission). PlySs2 wasPCR-cloned from genomic DNA from strain 89/1591 with the followingprimers: AATGCTAGCCTGATACACAGTTAGAGACC-fwd (SEQ ID NO:9) andCCTAAGCTTCTTTTCACAAATCATAATCCCCAG-rev (SEQ ID NO:10). The primersinclude restriction sites (NheI and HindIII) for cloning into pBAD24.The forward primer corresponds to a position ≈60 bp upstream of the genestarting point, because several in-frame ATG triplets are situated nearone another at the 5′-end. This enables the native ribosomal bindingsite (instead of the engineered RBS of pBAD24) to guide transcription.PlySs2 was cloned out of a prophage genome in S. suis into a pBAD24vector (pBAD24_PlySs2, FIG. 5) and transformed it into Escherichia coliTop 10 cells. pBAD24 encodes β-lactamase, enables tight transcriptionalcontrol, and is induced by inexpensive arabinose. The vector-transformedE. coli were grown on opaque plate containing Pseudomonas peptidoglycanhalos suspended in soft agar (Wang, Y et al (2009) Curr Microbiol58(6):609-615). Clearing zones appeared around the E. coli coloniesindicating expression of active PlySs2, which hydrolyzed thepeptidoglycan within the soft agar. The structure of PlySs2 is quiteunlike that of LySMP. It encodes a predicted N-terminal CHAP domain(cysteine-histidine amidohydrolase/peptidase, PF05257) and a C-terminalSH3-type 5 domain (PF08460) (FIG. 4). N-terminal sequencing confirmedthe start as “MTTVNEA . . . ”

Lysin Protein Production

E. coli containing the pBAD24_PlySs2 plasmid were grown at 37° C. in 10L of LB AMP100 and induced for overnight expression with 0.2% arabinoseat an OD₆₀₀-0.8. The cultures were spun at 10,722 rcf for 20 mins. Thepellets were resuspended in 100 mL of 15 mM Na₃PO₄, pH 7.4 and mixedwith protease inhibitor cocktail tablets. This mixture was homogenized,and the homogenate was centrifuged at 1,723 rcf for 20 mins. Thesupernatant was ultra-centrifuged at 30,000 rpm for 1 hr. Enough 15 mMNa₃PO₄, pH 8.0 was added to the supernatant to bring the pH to 7.4.

The protein was run over an anionic HiTrap Fast Flow DEAE column (15 mMNa₃PO₄ (PB), pH 7.4) without PlySs2 binding (FIG. 6A). Ammonium sulphatewas added to the flow through to a 40% concentration. The precipitatewas centrifuged and resuspended in 200 mL 15 mM Na₃PO₄, pH 6.7. Theprotein was dialyzed overnight in 15 mM Na₃PO₄, pH 6.7 with 20 μmtubing. The dialysate was run over a cationic HiTrap Fast Flow CM columnwith PlySs2 eluting cleanly in the shoulder of the flow through as wellas at 70 mM NaCl, 15 mM Na₃PO₄, pH 6.7 (FIG. 6B). All fractions showingpure PlySs2 were pooled (FIG. 6C). It is notable that there are threestart codons in frame proceeding PlySs2:“ATGATGCGTGGAAAGGAGAAGCCTATGACAACAGTAAATGAAGCATTA . . . ” (correspondingto: “MMRGKEKPMT TVNEAL . . . ”). A pure sample of the protein wassubmitted for protein sequencing to confirm c the start to be “MTTVNEAL. . . ”.

To express PlySs1, the clone was grown in Power Broth+LB-Booster (AthenaEnzyme System) to OD₆₀₀≈1.0 and induced with 0.2% arabinose. The culturewas shaken for 4 hr at 37° C. (inclusion bodies would form at longertimes). The expressing cells were pelleted, resuspended in 15 mMphosphate buffer pH 6.2, and lysed by three passages through anEmulsiFlex C-5 homogenizer. Residual debris was removed bycentrifugation (1 hr, 35,000×G), and ammonium sulfate was added at 225g/L (40% saturation). The precipitated protein was pelleted andresolubilized in 15 mM phosphate pH 7.4, and dialyzed against thisbuffer overnight. The dialysate was next passed through a DEAEanion-exchange column equilibrated against the same buffer (fast flowresin, General Electric).

The aforementioned preparation led to a highly pure lysin preparation injust two chromatographic steps (FIG. 6). With a predicted pi of 9.01,PlySs2 flowed directly through the DEAE column at pH=7.4 leaving thebulk of the contaminant proteins stuck to the DEAE column. PlySs2 elutedcleanly in the shoulder of the flow through and at 17 mM NaCl, beingpurified from proteins that rapidly flowed through the CM resin. Thispreparation yielded ≈60 mg of protein per liter of E. coli culture at≈1.5 mg/ml with >99% purity. The yield increased to ≈150 mg per liter ofE. coli culture at ≈2.0 mg/ml with >90% purity when the CM column stepwas forwent. If necessary, the latter product was: dialyzed (into 5 mMPB, 15 mM NaCl); lyophilized; reconstituted (at 10% of the originalvolume); centrifuged; and filter-sterilized (to remove any insolublematerial). This generated a soluble solution of PlySs2 at ≈20 mg/ml,which retained the concentration-adjusted activity of the lowerconcentration starting material. PlySs2 can be produced moreefficiently, and at a higher concentration, than many published lysins(Daniel A et al (2010) Antimicrob Agents Chemother 54(4):1602-1612;Wang, Y. et al (2009) Curr Microbiol 58(6):609-615; Nelson, D et al(2006) Proc Natl Acad Sci USA 103(28):10765-10770).

Biochemical Characterization of PlySs2

The S. suis lysins were further characterized and tested to determinebiochemical conditions including optimal pH, optimal salinity,temperature stability, and the effect of EDTA. The activity wasdetermined by the degree of S. suis 7997 turbidity reduction (OD₆₀₀)following the addition of PlySs2 at 32 μg/ml. Briefly, a 5 mL brainheart infusion (BHI) S. suis 7997 overnight culture was inoculated into45 mL BHI and grown at 37° C. for 2 hours. The 50 mL culture was spun at1,789 rcf for 10 min. A 50 mL culture of S. suis 7997 was centrifuged.The pellet was washed with 50 mL double-distilled H₂O (ddH₂O) for the pHtest, or 25 mL 15 mM Na₃PO₄, pH 8.0 for the other tests and centrifugedagain. The pellet was then resuspended in enough dd H₂O or 15 mM Na₃PO₄,pH 8.0 to bring the final OD₆₀₀ to ˜1.0 for each test condition. In allcontrols, PB replaced PlySs2. Spectrophotometric readings were taken ofeach sample at OD₆₀₀ every minute over an hour. The overall results foroptimal pH, optimal salinity, temperature stability and effect of EDTAare depicted in FIG. 7A-7D. The pH-dependence of the enzyme was firstaddressed using two buffer sets with adjacent pH ranges,citrate/phosphate: 4.6-8.0; and bis-tri-propane (BTP): 7.0-9.7. NaCl,EDTA, and DTT were also varied to test conditions for PlySs2 activity.To determine optimal pH, PlySs2 activity was tested against S. suisstrain 7997 in phosphate/citrate buffer at various pH levels (FIG. 7A).PlySs2 had the strongest activity at pH 8.0. We observed an extendedspectrum of lysis at the highest pH values. Optimal pH was similarlydetermined against S suis strain 7997 this time using Bis-tris propane(BTP) buffer, which permitted assessment up to a higher pH level (FIG.8). PlySs2 was shown to have acute activity up to a pH of 9.7. In BTP,lysis was maximal at the highest pH, 9.7, but this is not a suitablebuffer for living cells. Lysis also occurred in BTP, pH 7.0-8.0; atcommensurate pH-values, however, the magnitude of the OD-drop was muchmore pronounced in citrate/phosphate (a more physiological buffer forthe growth of test cultures). There was activity down to pH 6.0, whichis significant, because the pH of blood is approximately 7.4. Todetermine the optimal salt concentration, in 195 μl of cells, 5 μl lysinwere added to 50 μl of various NaCl concentrations (FIG. 7B). PlySs2 hadthe greatest activity in 0 mM NaCl. The cells are more susceptible tolysis within a more hypotonic solution. Salt did not enhancePlySs2-induced lysis. At constant enzyme concentrations, bacteriolysisdecreased from 0-1000 mM NaCl. Therefore, 0 mM NaCl is optimal, becausecells are more susceptible to lysis within a more hypotonic solution.

To determine the temperature stability of lysin, it was incubated for 30minutes at various temperatures, cooled and then added to 245 μl cellssuspended in 15 mM Na₃PO₄, pH 8.0 (FIG. 7C). Exposure of PlySs2 to anexcess of DTT had no impact (either positive or negative) on activity(data not shown). Treatment with EDTA impeded PlySs2-induced lysis of S.suis. Lysin was added to cells suspended in 15 mM Na₃PO₄, pH 8.0 alongwith various concentrations of ethylenediaminetetraacetate (EDTA) todetermine if it requires a cofactor. In controls, dd H₂O replaced lysine(PlySs2) for all tests. Very low concentrations ofethylenediaminetetraacetate (EDTA) diminish PlySs2 activity (FIG. 7D).This signifies that PlySs2 requires a cofactor or some other modifier.Lysin (PlySs2) was tested with EDTA at very low concentrations todetermine what level would allow some residual activity. At that level(between 4 uM and 200 uM EDTA), low (5-50 uM) amounts of differentdivalent cations (Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Zn²⁺) are added todetermine the cofactor.

The stability of PlySs2 was tested when incubated at: differenttemperatures for 30 minutes; 37° C. for hours; 4° C. for days; and −80°C. for months. The activity of each aliquot (at 32 μg/ml) against S.suis 7997 was spectrophotometrically determined as outlined above.PlySs2 was also tested after one to ten consecutive room temperature to−80° C. freeze-thaws. When incubated at 22° C.-85° C. for 30 min, PlySs2activity was principally unaffected through a significant temperaturerange, including 55° C.; at 60° C., PlySs2 activity was completelyabolished. After incubation at 37° C. for 24 hours, PlySs2 retained fullactivity; but after a 48-hour 37° C. incubation, PlySs2 showeddiminished activity. There was no observable decrease in activity after15 days of 4° C. incubation. In addition, PlySs2 lasted over 7 months at−80° C. without a reduction in activity. The lysin can endure 10consecutive room temperature to −80° C. freeze-thaws without anyobservable effect on its activity. The stability of purified PlySs2lysin was determined upon maintenance at 37° C. for up to 48 hours inbuffer. Killing effectiveness was determined against S. suis strain 7997periodically, as shown in FIG. 9. The PlySs2 lysin is >90% stable up to24 hours and maintains at least 50% activity after 48 hours Stability ofthe PlySS2 lysin was evaluated on freezer storage at −80° C. The PlySs2lysin retains essentially 100% activity on storage in buffer for up toat least 7 months at −80° C. (FIG. 10).

Investigations to determine the bond catalyzed by PlySs2 lysin have beenundertaken. PlySs2 was incubated with purified S. suis peptidoglycanstripped of lipotechoic acid and carbohydrates overnight at 37° C. andthe product submitted for mass spectroscopy. Data suggest that thecleavage is an N-acetylmuramoyl, -L-alanine amidase.

Biochemical Characterization of PlySs1

A prophage lytic enzyme was cloned from a functional genomic screen ofS. suis strain 7711, a serotype 7 isolate originating from theNetherlands1. The complete PlySs1 lysin gene encodes a 452-residueprotein: Pfam analysis predicts a type 5 alanine-amidase domain(PF05832) at the N-terminus, followed by a double CPL-7 cell-wallbinding domain (PF08230) in the central region, and a secondaryglucosaminidase domain (PF01832) at the C-terminus. Architecturally, thedomain arrangement of the cloned lysin is highly atypical. Gram-positivelysins typically consist of an N-terminal enzymatic domain and aC-terminal binding domain. While occasionally lysins are seen with twoN-terminal lytic domains, it is rare for a second enzymatic functionallyto be encoded after the binding domain. One example is the LambdaSa2lysin of S. agalactiae (Pritchard D G et al (2007) Appl EnvironMicrobiol 73(22):7150-7154). Working with LambdaSa2, Donovan andFoster-Frey surprisingly observed increased enzymatic activity followingremoval of the C-terminal glucosaminidase domain (Donovan D M andFoster-Frey J (2008) FEMS Microbiol Lett 287(1):22-33). With thismotivation, we engineered a truncated construct of the cloned lysin withonly the N-terminal enzymatic and central binding domains. Thistruncated construct was expressed and purified for subsequent functionalanalysis; activity and characterization studies described herein werebased on the truncated PlySS1; herein it is referred to as truncatedPlySs1 or ΔPlySs1. As above noted, the structure and amino acid sequenceof the full length and truncated PlySs1 lysin is depicted in FIG. 3.

The optimal biochemical conditions for PlySs1 were determined againstlive cells of the encoding S. suis strain (7711). For these experiments,activity was gauged through the degree of turbidity reduction (OD₆₀₀) ofan aqueous bacterial suspension following the addition of lysin. ThepH-dependence of the enzyme was first addressed using two buffer setswith adjacent pH ranges, citrate/phosphate: 4.6-8.0; andbis-tris-propane (BTP): 7.0-9.7. An extended spectrum of lysis wasobserved, from 5.4-9.4 (FIG. 11A). In BTP, lysis was maximal from8.2-9.0; at commensurate pH-values, however, the magnitude of theOD-drop was slightly more pronounced in citrate/phosphate (FIG. 11B).

The role of salt concentration was likewise considered, although it didnot greatly affect PlySs1-induced lysis. At constant enzymeconcentrations, bacteriolysis varied little from 0-1000 mM NaCl, withonly small numeric increases under the most hypotonic conditions (FIG.12). Exposure of PlySs1 to an excess of DTT or EDTA did not negativelyimpact activity, indicating that the enzyme does not rely uponintramolecular disulfide bridges or chelatable cations as cofactors(FIGS. 13A and 13B). The thermal stability of PlySs1 was examined byincubating the enzyme at various elevated temperatures prior to use (theOD-drop experiment itself was always conducted at 37° C.). When held at35° C.-60° C. for 30 min, lysin activity was virtually unaffected until50° C., at which point it was completely abolished (FIG. 14A). For the6-hr incubation, a partial decrease in activity was observed at 45° C.,while the 40° C. sample was unaffected (FIG. 14B). The lattercorresponds to typical porcine body temperature.

To determine the bond specificity of the enzymatic domain of PlySs1,purified S. suis cell walls (from type strain S735) were subject todouble digestion with HEWL (a muramidase) and PlySs1. The twopredominant peaks were m/z=718 and m/z=734. This corresponds exactly tothe predicted masses of the [Na-M]+ and [K-M]+ adducts ofGlcNAc-MurNAc-LAla-D-Gln. This suggests that PlySs1 possessesgamma-endopeptidase activity, cleaving the peptidoglycan stem betweenD-Gln and L-Lys as characteristic of a γ-D-glutaminyl-L-lysineendopeptidase. When a mass spectrum was taken of undigested cell wall,the above two peaks were absent.

Example 2 In Vitro Testing of Lysin Specific Activity PlySs2 Activity

To determine PlySs2 lysin activity against different cell types, 5 μL of1.6 μg/μL (8 μg) of PlySs2 was added in a microtiter well to 245 μL ofcells (suspended in 15 mM Na₃PO₄, pH 8.0). In a corresponding well ascontrol, 5 μL dd H₂O was added to 245 μL of cells. Readings (OD₆₀₀) weretaken for each well in a spectrophotometer every minute over an hour.The OD density indicates the amount of bacterial cell growth in themicrotiter well.

This activity test was first determined for the pathogenic S. suisstrain 7997 with various concentrations of PlySs2 (FIG. 15A). Specificactivity of purified PlySs2 lysin was also assessed in vitro against S.suis strain S735 (FIG. 15B). This test was then performed using 32 ug/mLPlySs2 to determine PlySs2 activity against other species of bacteria(it was found that based on lytic assay this was a good concentrationfor killing studies in vitro against other organisms) (FIG. 16A through16D). Further strain killing results are shown in FIGS. 17A and 17B.Additional results are tabulated below in TABLE 1.

As demonstrated and depicted in the above results, the PlySs2 lysinenzyme has broad activity killing not only against S. suis, but otherpathogens particularly including S. aureus, S. pyogenes, Listeria andGroup B streptococci. The results shown demonstrate reduction in growthand killing of methicillin resistant Staphylococcus aureus strains(MRSA). In comparable in vitro tests, PlySs2 is additionally andsimilarly effective against vancomycin intermediate sensitivityStaphylococcus aureus (VISA) and vancomycin resistant Staphylococcusaureus (VRSA) strains (FIG. 18).

This S. suis lysin is similar to previously identified and characterizedlysins in its ability to kill pathogenic bacteria quickly. However, itis unusual and remarkable in its broad activity against major pathogens.It is also notable that the lysin can be produced and purified readily,as shown above, and is stable in various relevant temperatures, pH andsalinity, making it an attractive candidate therapeutic enzyme.

TABLE 1 PlySs2 Reduction in Growth (Optical Density) of DifferentBacteria None Slight Moderate Acute (0.3-0.8 drop (0.05-0.3 drop(0.3-0.8 drop (>0.8 drop in OD₆₀₀) in OD₆₀₀) in OD₆₀₀) in OD₆₀₀)Bacillus Streptococcus Enterococcus Streptococcus suis, thuringiensissobrinus faecalis Strain (Serotypes): 10 (2), 735 (2) 6112 (1), 6388(1), 7997 (9), 8067 (9) Bacillus Streptococcus StreptococcusStaphylococcus cereus rattus dysgalactiae - epidermidis GGS BacillusStreptococcus Staphylococcus subtilis agalactiae - simulans GBS - 090RBacillus Streptococcus Staphylococcus anthracis pyogenes - aureus GASEscherichia Streptococcus Lysteria coli agalactiae - monocytogenes GBS -Type II Enterococcus faecium

PlySs1 Activity

Truncated PlySs1(ΔPlySs1) lysin activity was determined againstdifferent cell types. Given the above experiments, the following optimalbuffering conditions were employed for all further in vitro experimentswith ΔPlySs1: 20 mM phosphate buffer, pH=7.8, 2 mM EDTA. A range oflysin concentrations, from 6.5-130 μg/ml, were introduced to live S.suis cells in this buffer. Three strains were considered particularlyrelevant: 7711, the serotype 7 strain that encodes PlySs1; S735, theserotype 2 reference strain; and 7997, a highly virulent serotype 9strain. For each of these strains, the time-dependent OD600 response atvarious PlySs1 dosages is given in FIG. 19. In terms of bacterialviability, only the highest PlySs1-concentration (130 μg/ml) led toa >90% decrease in CFUs for 7711, S735, and 7997 after 1 hr treatment(TABLE 2). The lysin was also tested against actively-dividing cells inbroth culture (strain 7711) (FIG. 20). Although it delayed bacterialproliferation in a dose-dependent manner, these effects were generallymild and ΔPlySs1 could not inhibit S. suis growth outright.

TABLE 2 CFU Analysis of Strains 7711, S735 and 7997 Strain 13 μg/ml 130μg/ml S735 (ST2) 80.4%-92.6% 95.4%-99.5% 7997 (ST9) 16.8%-30.3%89.9%-93.9% 7711 (ST7)   0%-35.6% 95.3%-99.2%For two ΔPlySs1 concentrations (130 and 13 μg/ml), CFU analysis wasconducted on S. suis strains S735, 7997, and 7711 after 1 hr treatment(optimal buffering conditions). In each experiment, thepercentage-decrease in CFUs was determined for the treated sample versusthe untreated. The range of the values observed (across 3 independentexperiments) is reported here for each strain. The serotype of eachstrain is indicated in parentheses.

ΔPlySs1 was further tested against a panel of 19 other S. suis strainsof diverse serotypes, as well as other species of Gram-positivebacteria. The same lysin concentrations were used as above. For eachdosage, the observed lysis values after 1 hr are listed in TABLE 3 andTABLE 4, and the information is summarized graphically in FIG. 21.

TABLE 3 Analysis of Other S. Suis Strains 6.5 13 30 65 130 Strain μg/mlμg/ml μg/ml μg/ml μg/ml ST13 0.32 0.17 0.04 0.02 0.02 6112 (ST1) 0.140.11 0.06 0.02 0.01 ST8 0.25 0.12 0.06 0.03 0.03 6388 (ST1) 0.15 0.130.06 0.03 0.02 10 (ST2) 0.29 0.18 0.10 0.05 0.02 8076 (ST9) 0.52 0.400.21 0.14 0.04 ST9 0.50 0.30 0.23 0.13 0.05 ST4 0.63 0.47 0.32 0.22 0.12ST11 0.64 0.47 0.32 0.19 0.07 ST14 0.79 0.57 0.33 0.15 0.06 ST7 0.650.47 0.34 0.22 0.11 ST1 0.80 0.34 0.36 0.19 0.06 ST5 0.78 0.59 0.39 0.220.10 7197 (ST7) 0.64 0.49 0.39 0.16 0.07 ST6 0.76 0.56 0.40 0.21 0.06ST3 0.81 0.71 0.48 0.32 0.16 ST2 0.79 0.70 0.49 0.34 0.17 ST10 0.85 0.720.55 0.44 0.28 ST12 See Caption Below** **Various isolates of S. suiswere exposed (at optimal buffering conditions) to ΔPlySs1 at the aboveconcentrations. The majority of these bacteria are unnamed clinicalisolates of the indicated serotype (e.g. ST1, ST2, etc . . .). For thenamed strains, the serotype is given in parentheses. The 1-hourtreated/untreated OD₆₀₀- ratio is given for each Δ PlySs1 concentration(representing a single experiment), and the strains are listed in theorder of decreasing sensitivity. For strain ST12, it was not possible toconduct OD analysis. Upon the addition of ΔPlySs1 (all aboveconcentrations), the cells would rapidly self-adhere and fall out ofsuspension. This phenomenon was not observed for untreated ST12-cells.

TABLE 4 Analysis of Other Gram Positive Bacteria 6.5 13 30 65 130 Strainμg/ml μg/ml μg/ml μg/ml μg/ml S. oralis 35037 0.30 0.13 0.08 0.07 0.04S. agalactiae type II 0.61 0.21 0.11 0.08 0.04 S. dysgalactiae 215970.26 0.18 0.12 0.10 0.09 S. pyogenes A486 0.12 0.13 0.13 0.11 0.10 S.pneumoniae R36 0.25 0.22 0.14 0.16 0.12 S. dysgalactiae GGS 0.30 0.270.15 0.11 0.14 S. equi 700400 0.48 0.25 0.15 0.07 0.09 S. uberis 275980.42 0.23 0.16 0.14 0.12 S. pyogenes D471 0.39 0.27 0.17 0.13 0.09 S.gordonii 10558 0.76 0.32 0.19 0.09 0.06 S. equi 9528 0.66 0.45 0.25 0.190.16 L. monocytogenes HER1084 0.63 0.52 0.26 0.14 0.04 S. sanguinis10556 0.48 0.44 0.28 0.21 0.11 Group E streptococci K131 0.69 0.50 0.330.22 0.15 S. sobrinus 6715 0.64 0.48 0.39 0.32 0.23 E. faecium EFSK20.85 0.67 0.52 0.32 0.13 S. aureus RN4220 0.89 0.78 0.55 0.31 0.10 S.salivarius 9222 0.80 0.76 0.56 0.53 0.37 S. rattus BHT 0.82 0.84 0.820.83 0.79 M. luteus 4698 0.84 0.90 0.83 0.87 0.82 E. faecalis V583 0.980.93 0.84 0.71 0.52 B. cereus 14579 0.93 0.92 0.86 0.90 0.86 B.thuringiensis HD73 0.99 0.98 0.93 0.86 0.60 S. mutans U159 0.95 0.990.94 0.76 0.85 S. epidermidis HN1292 1.04 1.00 0.96 0.94 0.87 S.agalactiae 090R 0.97 0.99 0.97 0.98 0.93 S. simulans TNK3 0.96 1.00 1.001.00 0.96 B. anthracis ΔSterne 1.02 1.03 1.02 0.98 0.90 B. subtilis SL41.07 1.05 1.04 1.03 0.96

All S. suis strains demonstrated some degree of susceptibility.Interestingly, many of the non-suis streptococci (and even somenonstreptococci) also lysed at commensurate enzyme concentrations. Asdemonstrated and depicted in the above results, the PlySs1 lysin enzymehas broad and equivalent activity killing not only against S. suis, butagainst numerous Streptococcus strains, including Group B streptococci,and additionally against other pathogens, particularly including S.aureus, Enterococcus, Bacillus and Listeria. Classically, a phage lysindemonstrates a marked decrease in activity when going from within itshost species to outside of it. Here, however, a broad range ofsusceptibility was seen among non-suis bacteria, with some demonstratingidentical lysis to S. suis itself.

Example 3 CFU Killing Assay

The specific killing and drop in colony forming units (CFU's) of S. suisS735 and 7997 was determined when exposed to 32 ug/mL PlySs2 for 60minutes in 15 mM PB, pH 8.0 (FIG. 22).

Example 4 Assessment of Resistance

To test for the development of resistance to the S. suis lysin insusceptible bacteria, each of the Staphylococcal S. aureus strains MW2and 8325 and Streptococcus pyogenes strain 5005 were exposed toincrementally increasing concentrations of PlySs2. Neither S. aureusstrain developed resistance during the course of the study (FIG. 23).Following an established protocol (Rouse, M. S. et al (2005) AntimicrobAgents Chemother 49(8):3187-3191; Pastagia M et al (2011) AntimicrobAgents Chemother 55(2):738-44) for developing mupirocin-resistantstrains, S. aureus strains MW2 and 8325 and S. pyogenes 5005 strain weregrown in the presence of PlySs2. The concentration of PlySs2 doubleddaily from 1/32^(nd) of PlySs2's minimally inhibitory concentration(MIC) against each strain to 4× its MIC over an 8-day period. Initially,bacterial cells at 5×10⁸ CFU/ml were grown overnight in the presence of1/32×MIC PlySs2 in MHB at 37° C. These cells were centrifuged for 10 minat 900 rcf and divided into two aliquots. One aliquot was diluted10-fold into fresh MHB media with double the previous concentration ofPlySs2; a portion of the other aliquot was spread onto the surface of aMHA plate containing the MIC of PlySs2 to screen for resistant clones.Separate cultures of each strain were grown in the presence of mupiricinin the same manner as a positive control.

In this experiment, the MICs were determined by detection of pelletformation in the bottom of rounded polysterene plate wells. Each day,1.0 μL sample from each culture was spread on selective platescontaining the MIC of the respective drug to which each culture wasbeing exposed. The MIC of PlySs2 or mupirocin was tested for 4 coloniesper culture every day by microdilution for each serial passage asdescribed above (Wiegard, I et al (2008) Nat Protoc 3(2):163-175) todetermine if a resistant (defined as a 4-fold increase in MIC) clone hademerged. The procedure was repeated with mupirocin and each strain as apositive control.

Example 6 Oral Cavity Microbiota Study

The effects of the S. suis lysins on natural bacterial flora wereassessed using a rat oral cavity microbiota study. Blood agar plateswere streaked with swabs from the oral cavities of two rats. Cultureswere isolated from each plate through two cycles of passage and grownovernight in BHI broth. The next day, 1 mL of each culture was platedonto dry BHI agar plates resulting in a lawn of these cultures on agar.After they dried, 10 μL of PlySs2 was deposited on either side of acentral 10 μL dd H₂O drop as a control. Of 6 cultures, a clearing zonearound the PlySs2 drops only appeared on one culture (data not shown).This culture was sent out and confirmed as S. aureus. The oral cavitiesof each of 3 rats from Harlan, 4 Charles River, and 2 separate rats fromCharles River were swabbed. Yellow colonies grew on each mannitol saltplate streaked with the swab from each rat indicating that they allorally contained S. aureus (data not shown).

Example 7 MRSA Mouse Sepsis Model

PlySs2 lysin activity against S. aureus has been further evaluated in anMRSA mouse sepsis model. In the MRSA mouse sepsis model, susceptiblemice (FVB/NJ mice, weight range 15-20 g) were injected with 500 μL of5×10⁵ (approx LD₁₀₀) MRSA cells/mL in 5% hog gastric mucin in PBSintraperitoneally (IP). After 3 hours, all of the mice are bacteremicwith MRSA in their organs (including spleen, liver, kidney, andheart/blood). To determine PlySs2 activity in this model, 500 μL PlySs2at 2 mg/mL was injected IP 3 hours after injecting MRSA. This results in˜1 mg/mL PlySs2 within each test mouse's blood stream. Control mice wereinjected with 500 μL PBS. Survival was evaluated over ten days. Thecontrol mice die within 18-20 hours. This study yielded promisingresults, showing remarkable survival of mice treated with PlySs2 (FIG.25).

Example 8 Wound Infection Model

The S. suis lysin was tested in an MRSA wound infection model in rats.In this model, a 3 centimeter incision is created along the dorsal sideof the rat. Subsequently, a 1 cm long, 1 cm deep incision is cut intothe spinotrapezius muscle. 50 μL of MRSA at 1×10⁸ CFU's/mL (5×10⁶ CFU'stotal) is then inoculated into the wound. After 3-5 minutes, the testrats are treated with 500 μL of PlySs2 at 10 mg/mL. After 3-5 moreminutes, the wound is stapled shut and 100 μL of PlySs2 added at 10mg/mL to the outside of the wound. Care is taken to ensure that none ofthe bacteria nor the lysin escape the wound as it is sealed off withstaples. After 10 days, the CFUs of MRSA are examined within theinfections site. In the first 2 rounds of tests PlySs2 was shown to havea deleterious effect on MRSA in the dorsal wounds, with the CFU's ofMRSA dropping 3-4 logs in the PlySs2 treated rats compared with thecontrol rats.

Animal Experiments

Animal experiments were initiated by determining the infectious dose ofMRSA necessary to cause infection in the rat wound. We found that evenat doses up to 10⁹ CFUs no infection occurred. However, when we added aforeign body (sterile glass beads), infection occurred at <10⁸ CFUs.Specifically, incisions were made in the backs of the rats (5-6 cm inlength) and a 2-3 mm incision was made in the underlying tissue. To thiswas added 100 mg sterile sand and 50 μl of MRSA. The wounds were stapledclosed and the animals followed for 10 days and the wounds opened andexamined for gross changes and tissue samples taken for bacteriologicalexamination.

Results

Control animals that received only sterile sand showed normal healingwith no unusual characteristics and no bacterial contamination. Animalsthat received MRSA showed clear abscess formation, necrotic tissue andpus. Samples (weighed and homogenized tissue) taken from the openedwound yielded about 10⁷ CFU MRSA/gram of tissue. This model was veryreproducible yielding the same results in at least 10 animals.

Treatment

We used the model as described above to treat with PlySs2 lysin todetermine the effects of the treatment. In this case, half of theanimals receiving the MRSA and sand were treated with 10 mg PlySs2 in 50ul of phosphate buffer (PB), control animals received PB instead 10minutes after MRSA dosing. The animals were followed for 10 days. Atthis time the animals were analyzed for gross changes and microbiology.The wounds in control animals exhibit puss, necrosis, and poor healing.This is in sharp contrast to animals treated with a single dose ofPlySS2. These wounds did not exhibit pus or necrosis and exhibitedbetter healing.

Results

As can be seen in TABLE 5, the wounds of rats that were treated withbuffer alone exhibited an average of 4.27×10⁶ CFU/gram of tissue of MRSAwhile animals treated with PlySs2 had an average of 1.41×10² CFU/gram oftissue, a reduction of >4-logs of MRSA. This number is lower than 4-logssince most of the PlySs2-treated wounds were lower than our detectiblelimits.

TABLE 5 Rat MRSA Wound Infections After Treatment With PlySS2 or BufferPLYSS2 CFU/Gram Buffer CFU/Gram P1 <5.00E+01 B1 1.80E+05 P2 <5.00E+01 B21.18E+06 P3  3.20E+02 B3 2.05E+05 P4  3.20E+02 B4 7.25E+06 P5 <5.00E+01B5 3.20E+05 P6 <5.00E+01 B6 1.40E+07 P7  9.10E+01 B7 5.80E+05 P8<5.00E+01 B8 3.00E+05 P9  5.00E+01 B9 9.60E+06 P10 <5.00E+01 B104.40E+06 P11 <5.00E+01 B11 2.94E+06 P12  8.40E+02 B12 4.81E+06 P13<5.00E+01 B13 5.83E+06 P14 <5.00E+01 B14 2.82E+06 P15 <5.00E+01 B159.63E+06 AVG*  1.41E+02 AVG 4.27E+06 CFU Reduction vs Control 3.02E+04<=CFU below level of detection No Growth on Plates *Not true Avg since <actual #

Example 19 In Vivo Nasal Decolonization of MRSA

Carriage of both MSSA and MRSA in the human anterior nares is the majorreservoir for S. aureus infection. Studies have shown that roughly 80%of the population could be nasally colonized by S. aureus, and thatcolonization can be an increased risk factor for developing other moreserious S. aureus infections (Kluytmans, J., A. van Belkum (1997) ClinMicrobiol Rev 10(3):505-520). In fact, assessment of nasal colonizationis being instituted on admission to critical care settings in hospitalsin the U.S. Elimination of nasal carriage in the community or in thehospital setting thus could possibly reduce the risk of infection andslow the spread of drug resistant S. aureus. To study the ability of S.suis lysin to reduce MRSA colonization of the nasal mucosa, C57BL/6Jmice are intranasally inoculated with ˜2×10⁷ of a spontaneouslystreptomycin resistant strain of MRSA (191-SMR). Twenty-four hourspost-infection mice are administered three doses (1 mg) hourly of eitherphosphate buffered saline (control), or PlySs lysin into the nasalpassages. One hour after the last treatment, mice are sacrificed andbacteria colonies enumerated on Spectra MRSA agar (a selectivechromogenic medium developed to diagnostically detect MRSA nasalcolonization) and Columbia blood agar. Three independent experiments areperformed to evaluate at least 10 mice for each treatment group.Significantly reduction in the mean CFU on the nasal mucosa on treatmentwith S. suis lysin is determined.

REFERENCES

-   1. Beres, S. B., J. M. Musser. Contribution of Exogenous Genetic    Elements to the Group A Streptococcus Metagenome. PLoS ONE, 2007.    2(8):1-14.-   2. Cantin, M., J. Harel, R. Higgins, M. Gottschalk. Antimicrobial    resistance patterns and plasmid profiles of Streptococcus suis    isolates. Journal of Veterinary Diagnostic Investigation, 1992.    4:170-174.-   3. Fischetti, V. A. Bacteriophage lysins as effective    antibacterials. Current Opinion in Microbiology, 2008. 11:393-400.-   4. Nelson, D., L. Loomis, V. A. Fischetti. Prevention and    elimination of upper respiratory colonization of mice by group A    streptococci by using a bacteriophage lytic enzyme. Proceedings of    the National Academy of Sciences of the United States of    America, 2001. 98:4107-4112.-   5. Sriskandan, S., J. D. Slater, Invasive Disease and Toxic Shock    due to Zoonotic Streptococcus suis: An Emerging Infection in the    East? PLoS Medicine, 2006. 3(5):585-587.-   6. Wang, I. N., D. L. Smith, R. Young, Holins: the protein clocks of    bacteriophage infections. Annual Review of Microbiology, 2000.    54:799-825.

Example 20 Bacteriophage Lysin PlySs2 with Broad Lytic Activity ProtectsAgainst Mixed Methicillin-Resistant Staphyloccocus aureus andStreptococcus pyogenes Infection

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcuspyogenes (group A streptococci—GrAS) cause several infectious humandiseases. These bacterial pathogens are among the many Gram-positivepathogens that have established resistance to leading antibiotics. Thereis a need for alternative therapies to combat these infectious agents.We have developed a novel bacteriophage (phage) lysin with activityagainst MRSA, vancomycin-intermediate S. aureus, Streptococcus suis,Listeria, Staphylococcus simulans, Staphylococcus epidermidis,Streptococcus equi, Streptococcus agalactiae, S. pyogenes, Streptococcussanguinis, group G streptococci, group E streptococci, and S.pneumoniae. This phage lysin from S. suis was termed PlySs2. Consistentwith previous exogenous lysins, PlySs2 did not display activity againstany Gram-negative bacteria (eg. Escherichia, Bacillus, or Pseudomonas).PlySs2 has an N-terminal cysteine histidine aminopeptidase (CHAP)domain, and a C-terminal SH3b binding domain. PlySs2 is stable at 50° C.for 30 min, 37° C. for 24 hours, 4° C. for 15 days, and −80° C. for >8months. It maintains full activity after 10 freeze-thaws. At 128 μg/ml,PlySs2 was able to reduce colony forming units (CFUs) of MRSA and S.pyogenes by 5-logs and 3-logs, respectively. The minimum inhibitoryconcentration (MIC) of PlySs2 was 16 μg/ml for MRSA. A single 2 mg doseof PlySs2 protected 22 of 24 mice in a mouse septicemia model of a mixedMRSA and S. pyogenes infection. After serially increasing exposure toPlySs2, neither MRSA nor S. pyogenes established resistance to PlySs2 asMRSA did to mupirocin. No lysin has shown such effective broad lyticactivity; stability; and efficacy against leading human bacterialpathogens. PlySs2 is a promising therapeutic for MRSA, S. pyogenes, andmany other pathogens without incidence of resistance.

There are many Gram-positive pathogens causing disease and infectionworldwide, including: S. pyogenes, S. aureus, S. agalactiae, Listeria,and others. They cause a variety of diseases, and there are limits tocurrent treatments.

Over 30% of the human population may be colonized with Streptococcuspyogenes in the upper respiratory tract—the only known site of benigncolonization [1]. Colonized individuals are much less likely thanseverely sick persons to transmit illness [1]. S. pyogenes (group Astreptococci—GrAS), annually infects over 750 million people [2-4]. Eachyear, there is a 25% mortality rate among the ≈650,000 cases thatprogress to severe infection [2]. S. pyogenes causes pharyngitis in theupper respiratory tract, and impetigo within the skin of human hosts[5]. Scarlet fever, erysipelas, cellulitis, necrotizing fasciitis, andtoxic-shock syndrome and other illnesses that emerge from S. pyogenesinfection. The mortality rates can be very high for these infections,including 20% for necrotizing fasciitis, and 50% for toxic-shocksyndrome [6]. Rheumatic fever, acute glomerulonephritis, and forms ofobsessive-compulsive disorder are non-suppurative sequelae associatedwith a S. pyogenes [7]. Rheumatic fever outbreaks have seen a riseworldwide since the 1980's [8]. Though rare, rheumatic fever canprogress to severe illness if it enters deep into soft tissue [4].

Of all the Gram-positive pathogens, Staphylococcus aureus has become themost difficult to treat. S. aureus is a Gram-positive facultativeanaerobe that causes most Staphylococcus infections in man. Humananterior nares (nostrils) are typically the primary sites of S. aureuscolonization, along with other moist openings on the body serving asadditional sites for entry [9-12]. S. aureus often causes severesecondary infections in immunocompromised individuals, as well ascausing disease in otherwise healthy individuals. In addition to skinand soft tissue infections (SSTIs), it can cause sepsis, toxic shocksyndrome, and necrotizing pneumonia, necrotizing fasciitis, andpyomyositis, endocarditis, and impetigo. These infections are usuallytreated with methicillin, mupirocin, or vancomycin.

Many S. aureus strains, such as methicillin-resistant S. aureus (MRSA)and vancomycin-resistant S. aureus (VRSA), have evolved resistance toone or more antibiotics used as standard treatment [13], making themeven more difficult to treat with available antimicrobials [13]. Thecausative pathogen for numerous nosocomial infections, MRSA accounts formore than 50% of S. aureus hospital isolates causing pneumonia andsepticemia [14]. Further exacerbating the problem, MRSA is readilytransmitted between patients in hospitals [15]. Nearly half of allbacteremia cases in intensive care units are caused by MRSA having amortality rate of 30-40% [16, 17]. It is the primary cause of lowerrespiratory tract infections, surgical site infections, and 19,000deaths/year in the US alone [14, 18].

While health-care-associated MRSA infects susceptible patients,community-associated MRSA (CA-MRSA) infections arise in healthyindividuals [19, 20]. CA-MRSA strains seem to be more virulent andcontagious than traditional MRSA strains in humans and animal models,causing more severe diseases [21-24]. Distinct strains of CA-MRSA areepidemic in Europe, North America, Oceania, and other regions [20, 25,26]. The MW2 strain (pulsed-field type USA400) is the prototypicalCA-MRSA, having contributed to the incipient outbreak of CA-MRSA in theUSA, thus leading to an epidemic [19, 27].

A beta-hemolytic Gram-positive streptococcus, Streptococcus agalactiae(Group B streptococci—GBS) contains an antiphagocytic capsule as itsprimary virulence factor [28, 29]. S. agalactiae can exist in the humangastrointestinal system, occasionally colonizing secondary sites likethe vagina in over 33% of women [30, 31]. The colonizing S. agalactiaecan infect a neonate during birth resulting in bacterial septicemia,making early-onset S. agalactiae the primary cause of death in newbornsfor over 4 decades [32-34],[35]. The current standard of practiceexposes the mother to antibiotics that further the likelihood ofresistance.

A recent Gram-positive pathogen outbreak involves Listeria, and haskilled 29 in the United States as from July to November 2011—the mostdeadly food-borne illness outbreak in the US since the 1970's [36]. Mostindividuals contract listeriosis after consumption of contaminated food,facing a mortality rate of 20-30%, even with antibiotic therapy [37,38]. Listeria survives well in food processing systems and the humangastrointestinal tract, readily adjusting to swift changes in pH,salinity, and temperature [37, 39, 40].

There are many other Gram-positive human pathogens, including:Streptococcus sanguinis (dental plaque and caries); S. sanguinis(endocarditis); Group G Streptococcus; Group E Streptococcus; and S.pneumococcus (pneumonia, otitis media, meningitis, bacteremia, sepsis,endocarditis, peritonitis, and cellulitis).

Zoonotic Gram-positive pathogens include: Streptococcus equi(strangles—an upper respiratory tract infection—in equines, eg. horses);and Streptococcus suis (sepsis and meningitis in pigs and humans). Thepathogenic S. suis serotype 9 strain 7997 has been associated withincreasing reports of zoonotic transmission from pigs to humans [41].Humans and pigs have been treated with penicillin or gentamicin, but S.suis isolates resistant to these antibiotics exist [42]. S. suis maydevelop a consistent presence in human populations in years to come.

There has been a sharp increase in antibiotic resistance among all ofthese Gram-positive microbes. Therefore, alternative therapies must bedeveloped to treat bacterial pathogens. Novel antimicrobial sourcesinclude enzyme-based antibiotics (“enzybiotics”) such as phage lyticenzymes (also known as endolysins or “lysins”).

Lysins have garnered much attention recently as novel antibacterialagents (reviewed in [18, 43, 44]). After bacteriophage viruses replicateinside of a host, their progeny must escape. Phage encode both holinsthat open a pore in the bacterial membrane, and peptidoglycan hydrolasescalled lysins that break bonds in the bacterial wall [45]. SinceGram-positive bacteria are hypotonic to their surroundings, disruptionof the cell wall leads to osmotic lysis of the bacteria and release ofviral progeny [18]. These peptidoglycan hydrolases (catalyzing a varietyof specific bonds) are encoded by virtually all double-stranded DNAphages.

Lysins can be cloned from viral prophage sequences within bacterialgenomes, recombinantly expressed in Escherichia coli, purified, and usedfor treatment [46]. When applied exogenously, these proteins are able toaccess the bonds of a Gram-positive bacterium's cell wall, as thepeptidoglycan of these species is continuous with the extracellularspace [18]. Lysins kill bacteria quicker than any known non-chemicalagent biological compounds [47-49].

Lysins have been shown to demonstrate a high lethal activity againstnumerous Gram-positive pathogens—generally, species that the encodingphage infects or closely-related organisms [18, 47]. Having beenproposed as potential enzybiotic agents, lysins are notable for thepotency and specificity they demonstrate toward particular bacteria [47,48, 50, 51]. As such, they should have a less dramatic affect on thenormal nonpathogenic flora in the host than broader-acting antibiotics[18]. To date, no lysin has shown broad in vivo activity againstmultiple species of bacterial pathogens.

Lysins have been developed against MRSA, including ClyS (astaphylococcal-specific chimeric lysin previously developed in our lab)[50]. Lysins have been evolved by viruses in an evolutionary struggle toinfect bacteria for billions of years. Therefore, there is selectivepressure for them to chose a feature that is essential to normalbacterial function—and thus, unlikely to be altered.

Two phages infecting S. suis have been previously isolated and studied.Harel et al. induced a siphoviral prophage from the genome of a serotype2 strain 89-999, although the identity of its lysin remains undetermined[52]. More recently, Ma and Lu isolated a lytic phage from nasal swabsof healthy pigs, sequencing its 36 kb genome [53]. This phage, termedSMP, demonstrated a limited host range, infecting only 2/24 S. suisstrains within serotype 2. The same group later PCR-cloned andrecombinantly expressed the SMP lysin (LySMP); the enzyme demonstratedbacteriolytic activity in vitro against several S. siuis serotypes.LySMP, as a recombinant protein, did not fold properly by itself and wasonly active in the presence of reducing agents, which may limit itspotential for in vivo trials [54].

There are over 11 completed S. suis genomes in the NCBI database. Thesequences of S. suis strains were analyzed to identify candidatepotential phage lysins within prophage regions and a new phage lysinfrom S. suis (termed PlySs2) was ultimately identified, isolated, clonedand characterized (see above Examples). Additional characterization isnow provided including the acute activity of PlySs2 against MRSA and S.pyogenes.

Materials and Methods

Bacterial Strains.

All strains were stored at −80° C. (TABLE 6). Staphylococcus,Streptococcus, Listeria, Enterococcus, Pseudomonas, Bacillus spp.strains were cultivated in brain heart infusion (BHI) medium.Escherichia coli was grown in Luria Bertain (LB) medium. All media wereacquired from Becton, Dickinson, and Company (Sparks, Md.), unlessotherwise stated. Bacteria were maintained at 37° C. Overnight cultureswere grown at 37° C., and shaken at 200 rpm, if necessary.

TABLE 6 Listing of Strains Organism Serotype Strain ATCC Source^(a)Notes Group E 2 K131 123191 1 Streptococcus Streptococcus suis 9 7997 6Streptococcus 6715 1 sobrinus Streptococcus 10556 1 sanguinisStreptococcus rattus BHT 1 Streptococcus M6 D471 1 pyogenesStreptococcus MΔ D471 1 mutant JRS75 pyogenes Streptococcus M6 10394 1pyogenes Streptococcus M49 NZ131 1 pyogenes Streptococcus M4 1streptomycin resistant - pyogenes mucoid Streptococcus M3 315 1 pyogenesStreptococcus M18 8232 1 pyogenes Streptococcus M1 SF370 1 phage 159-1KO - pyogenes mucoid Streptococcus M1 SF370 1 phage 159-1 KO pyogenesStreptococcus M1 SF370 1 mucoid pyogenes Streptococcus M1 SF370 1 mucoidpyogenes Streptococcus M1 SF370 1 pyogenes Streptococcus M1 5005 1pyogenes Streptococcus 9V DCC1335 1 pneumoniae Streptococcus 6 DCC1850 1pneumoniae Streptococcus 15 DCC1476 1 pneumoniae Streptococcus 11 1pneumoniae Streptococcus 1 mutant Lyt 4-4 pneumoniae Streptococcusoralis 35037 1 Streptococcus mutans U159 1 Streptococcus 10558 1gordonii Streptococcus equi 9528 1 Streptococcus equi 700400 1 zooStreptococcus 1 Group G Streptococcus dysgalactiae Streptococcus 26RP661 Group C Streptococcus dysgalactiae equisimilis Streptococcus Type II 1Group B Streptococcus agalactiae Streptococcus 090R 1 Group BStreptococcus agalactiae Staphylococcus 2 TNK3 simulans StaphylococcusHER 1292 3 epidermidis Staphylococcus 4 vancomycin aureus intermediateresistance IV Staphylococcus 4 vancomycin aureus intermediate resistanceIII Staphylococcus RN4220 1 aureus Staphylococcus Newman 2 methicillinsensitive - aureus mutant LyrA Staphylococcus Newman 2 methicillinsensitive aureus Staphylococcus MW2 5 methicillin resistant - aureuscommunity acquired Staphylococcus 192 1 methicillin resistant aureusStaphylococcus 1 methicillin resistant aureus from patient DSStaphylococcus 1 highly mupirocin aureus resistant Staphylococcus 1D712 - daptomycin aureus resistant Staphylococcus 1 0325 - daptomycinaureus resisitant Pseudomonas RS1 1 aeruginosa Listeria HER 1184 1monocytogenes Listeria 4b N3013 1 monocytogenes Listeria 3b FSLJ 1 1monocytogenes Listeria 1 RS823 monocytogenes Listeria 1 RS820monocytogenes Listeria HER1083 1 monocytogenes Listeria BAA-680 1monocytogenes Escherichia coli Top10 1 Enterococcus 1 EFSK-2 faeciumEnterococcus faecalis V583 1 Bacillus thuringiensis HD-73 1 Bacillussubtilis SL4 1 Bacillus cereus 14579 1 Bacillus anthracis Δ sterne 1^(a)1, The Rockefeller University Collection; 2, Olaf Schneewind,University of Chicago, Chicago, IL; 3, Barry Kreiswirth, Public HealthResearch Institute, New Jersey; 4, Alexander Tomasz, The RockefellerUniversity; 5, ATCC; 6, Jaap A. Wagenaar, Utrecht University, Utrecht,Netherlands.

CFU Studies.

Log-phase bacteria were resuspended in buffer A to an OD₆₀₀ of 0.1 (=0.5McFarland≈10⁸ CFU/ml). PlySs2 was added at 128 μg/ml to polypropylenemicrotiter plates (Costar) in triplicate for each test organism. Plateswere sealed and incubated at 37° C. with shaking every 5 minutes for 1hour. After 1 hour of incubation, cells were serially diluted in 10-foldincrements and plated on BHI. Triplicate controls for each strain wereperformed with buffer B replacing PlySs2.

MIC Studies.

The Wiegand, et al. protocol to determine minimum inhibitoryconcentrations was followed with adjustments detailed below [57]. Afinal suspension of ≈5×10⁵ cells/ml in MHB (or BHI for S. pyogenes)resulted after Sterile-filtered lysin or vehicle was added at theappropriate concentration [57]. These tests were distributed within a96-well microtiter plate. The MIC's were determined, in this experiment,by detection of pellet formation in the bottom of rounded polystereneplate wells. They were also corroborated colorimetrically withalamarBlue®.

In Vivo Murine Model.

The Rockefeller University's Institutional Animal Care and Use Committeeapproved all in vivo protocols. A systemic infection model described inDaniel, A. et al., was used to test for the in vivo efficacy of PlySs2against multiple gram-positive bacteria [50]. Briefly, 4-5 week oldfemale FVB/NJ mice (weight range 15 to 20 g) were obtained from TheJackson Laboratory (Bar Harbor, Me.). After a period of acclimation,mice were injected intraperitoneally (IP) with 0.5 ml of mid log-phase(OD₆₀₀ 0.5) bacteria diluted with 5% hog gastric mucin (Sigma) insaline. Bacterial suspensions contained ˜5×10⁵ CFU/ml of MW2, a PVLtoxin-encoding MRSA strain, ˜1×10⁷ of MGAS5005, an M1 serotype ofStreptococcus pyogenes that is virulent in humans and mice (Musser), ora combination of both bacteria simultaneously at the aboveconcentrations for the mix infection experiments. Actual bacterialinoculation titers were calculated by serial dilution and plating toColumbia blood agar plates for each experiment. Mice became bacteremicwithin one to three hours and contained MRSA and/or S. pyogenes in theirorgans (including spleen, liver, kidney, and heart/blood) ([50], andunpublished observations). Three hours post-infection the animals weredivided into 4 to 5 treatment groups and were intraperitoneallyadministered 0.5 ml of either 20 mM phosphate buffer, 2 mg/ml of thestreptococcal lysin PlyC [59], 2 mg/ml of ClyS [50], 2-4 mg/ml PlySs2,or a combination of 2 mg/ml PlyC and 2 mg/ml of ClyS. The survival ratefor each experimental group was monitored every 12 hours for the first24 hours then every 24 hours up to 10 days post-infection. The data werestatistically analyzed by Kaplan Meier Survival curves and a Logranktest performed for 95% confidence intervals using the Prism computerprogram (GraphPad Software; La Jolla, Calif.).

Results

Broad Lytic Activity of PlySs2.

All tested strains of S. aureus were highly susceptible to PlySs2 lysis(FIG. 25). This includes strains resistant to methicillin, vancomycin,daptomycin, mupirocin, and lysostaphin. PlySs2 activity againstvancomycin-intermediate S. aureus (VISA) and Newman strains was lesssevere than against other strains, but nevertheless robust. In additionto aureus, other Staphylococcus species (epidermidis and simulans) weresensitive as well. PlySs2 exhibited conventional activity against itsnative species, S. suis. Two strains of Listeria exhibited significantlysis from PlySs2, but other Listeria strains were impervious to PlySs2treatment. To a lesser extent, PlySs2 had activity against S. equi zoo,S. equi, S. agalactiae Type II (encapsulated), and S. agalactiae 090R.Of note, PlySs2 also had activity against all strains of S. pyogenes.This included serotypes M1, M3, M4, M6, M18, M49, and a variant withoutM protein. Unencapsulated, capsulated, and mucoid strains S. pyogenesall displayed comparable susceptibility to PlySs2.

There were a number of species that displayed less lysis than thoseabove. They were Streptococcus sanguinis, group G Streptococcus, group EStreptococcus, Enterococcus faecalis, and one strain of S. pneumococcus.S. gordonii was the only commensal against which PlySs2 had substantialactivity. The outer membrane surrounding the Gram-negative peptidoglycanprevented PlySs2 from displaying activity against Escherichia, Bacillus,or Pseudomonas, as expected.

The activity of PlySs2 was compared to that of ClyS in a simultaneous,side-by-side test using the same batch of cells (data not shown). Theactivity of each was comparable, but PlySs2 is far more tractable; wecontinued to pursue PlySs2 as a therapeutic against MRSA. Classically, aphage lysin demonstrates a marked decrease in activity against specimensoutside of its host species. Here, however, a broad range ofsusceptibility was seen among non-S. suis bacteria, with somedemonstrating more sensitivity than S. suis.

Efficacy of PlySs2 Against Gram-Positive Pathogens.

PlySs2 displayed significant activity reducing the number of L.monocytogenes, S. agalactiae, S. aureus, and S. pyogenes (FIG. 26). Itreduced all tested strains of S. agalactiae and L. monocytogenes morethan S. pyogenes 5005. The negative control E. coli was not reduced innumber after PlySs2 treatment.

MIC of PlySs2 for Pathogens.

The MIC of PlySs2 was relatively low for L. monocytogenes and S. aureus(FIG. 27). S. pyogenes and S. agalactiae registered similar MICs ofPlySs2. The MIC for the negative control E. coli was too high tocalculate.

Murine Mixed MRSA and S. pyogenes Septicemia Model.

To determine if the broad lytic activity of PlySs2 could provide in vivoprotection from infection with gram-positive pathogens, either alone oras a mixed infection, FVB/NJ mice were intraperitoneally injected with˜5×10⁵ CFU of MRSA strain MW2, ˜1×10⁷ of S. pyogenes strain MGAS5005and/or both bacteria simultaneously at the concentrations above. Threehours later mice were divided into 5 treatment groups and injected IPwith either 20 mM phosphate buffer, 1 mg of the control staphylococcallysin; ClyS, 1 mg of control streptococcal lysin; PlyC, 1 mg of ClyS+1mg of PlyC, or PlySs2 (1 mg for MRSA infections or 2 mg forstreptococcal and mixed infections, respectively). Mice were thenmonitored for survival over ten days. The results from 4 independentexperiments were combined and mouse survival data plotted with a KaplanMeier Survival curve (FIG. 28).

Within twenty-four hours of MRSA infection alone 17/18 buffer controlmice and 10/12 PlyC streptococcal lysin treated mice died of bacterialsepsis throughout their organs (including spleen, liver, kidney, andheart/blood). Only 2/18 of PlySs2 treated mice died at forty-eighthours, the remaining PlySs2 treated mice survived over the 10-day courseof the experiments with mice results comparable to the staphylococcalspecific lysin: ClyS (24/28, 86%) versus PlySs2 (16/18, 88%) (FIG. 28A).

Mice infected with S. pyogenes alone tended to succumb at a slower rate,14/15 buffer treated mice and 12/12 Clys staphylococcal specific lysintreated mice were dead by day three and four, respectively. On thecontrary, only 1/16 PlySs2 mice died at day three, the rest survived(15/16, 94%) to give results comparable to the streptococcal specificlysin PlyC (12/12, 100%) (FIG. 28B). To simulate a mixed bacterialinfection, mice were injected IP with a mixture of both MRSA and S.pyogenes from the bacterial inoculums' above. Treatment with buffer orthe single specific lysin controls did not significantly prolong mousesurvival. A majority of the PlyC (15/18) and buffer (21/23) treated micedied within 24 and 48 hours, respectively (FIG. 28C). While the mixinfection animals treated with ClyS succumbed slower, with 14/16 dead byday 4, similar to mice infected with only S. pyogenes. In contrast tothe controls most of the PlySs2 treated mice survive the mixed infection(22/24, 92%) and was comparable if not better to the mice treated withboth Clys+PlyC at the same time (16/20, 80%) (FIG. 28C).

PlySs2 Demonstrates Rapid Kill Versus Antibiotics in MRSA Strains

Several MRSA strains (Strain 245, 223, 926 and 932), which alldemonstrate similar MIC values (approx 32 μg/ml) for PlySs2, were testedfor log kill by direct comparison between PlySs2 and vancomycinantibiotic (1 μg/ml). Cultures of about 5×10⁵ bacteria are grown in MHBmedia in 50 ml conical tubes at 37° C. shaking 225 rpms for up to 6hours in combination with PlySs2 alone, vanvomycin alone, or noadditive. At various time points (15 min, 30 min, 1 hr, 2 hrs, 3 hrs, 4hrs, 5 hrs and 6 hrs) aliquot samples (approx 300 μl) are removed. Thelytic enzyme or antibiotic are inactivated by addition of a charcoalsolution to all aliquot samples. Samples are diluted and plated on agarplates to determin viable bacterial cell counts. The Log reduction inCFU is depicted in FIG. 29. PlySs2 provides rapid and effective killwith enhanced activity versus standard-of-care antibiotic vancomycin onall MRSA strains tested and depicted.

Discussion

The novel, native S. suis lysin, PlySs2, is demonstrated herein todisplay broad lytic activity against multiple Gram-positive pathogensincluding S. pyogenes and S. aureus in vivo. This lysin is the first todisplay such promiscuous activity; all previously characterized lysinsdisplay activity against a narrow spectrum of species [48, 50, 61-64].ClyS, has been shown to clear septicemic MRSA infections [50], however,to date, no lysin has been shown to clear more than one septicemicinfection, and none has cleared a mixed infection of any kind. Theability of PlySs2 to clear a mixed infection of staphylococci andstreptococci comes from its broad lytic activity. Although the in vitroactivity of PlySs2 is more robust against MRSA than S. pyogenes, the invivo data provided herein demonstrates its efficacy as an effectivetherapeutic against both bacteria. This may be due to differences in thestructure or composition of the S. aureus cell wall compared to that ofS. pyogenes affecting substrate accessibility. All of the Gram-positivepathogens against which PlySs2 has activity have developed resistanceagainst conventional antibiotics. Neither S. pyogenes nor MRSA were ableto establish resistance to PlySs2.

A strength of many lysins (including PlySs2) is their specificity.Antibiotics act nonspecifically, killing commensal microbes along withthe target pathogen. This results in many negative side effects (eg.diarrhea) and antibiotics can enable opportunistic pathogens (eg.Clostridium difficile) to cause entirely new infections. Lysins can beused to treat a single pathogen without disrupting the entire bacterialflora [18]. The specificity of many previously described lysins can alsobe a limitation in that multiple lysins would be needed to treatmultiple pathogens. Our findings provide a single lysin that can be usedto treat many pathogens while retaining a degree of specificity. PlySs2is active against various Gram-positive pathogens leaving theGram-negatives unaffected.

PlySs2 can serve as a viable treatment for infections caused by S.aureus and/or S. pyogenes such as: scarlet fever; erysipelas;cellulitis; necrotizing fasciitis; endocarditis; bacteremia/sepsis; avariety of abscess and non-abscess forming SSTIs; and impetigo.Treatment against neonatal septicemia and food-borne illness is alsoapplicable, because it displays more in vitro activity against S.agalactiae and L. monocytogenes than it does against S. pyogenes 5005(FIG. 25-27).

As a lysin, PlySs2 rapidly kills its target, far quicker thanantibiotics (FIG. 29). PlySs2 only has to contact the bonds of theexternal cell wall to mediate its effect; conventional antibiotics mustremain at high concentrations for an extended period, resulting in amultitude of side effects. This feature has enabled phage lysins totreat systemic infections in the past [48, 61]. As demonstrated herein,PlySs2 was able to clear septicemic MRSA and S. pyogenes infections froma high percentage of mice using a single dose of PlySs2. In addition,mixed septicemic MRSA and S. pyogenes infections were cleared from ˜92%of mice with a single dose of PlySs2 compared to ˜4% clearance forcontrols. Further enhanced kill or clearance may arise from increased orrepeated dosage.

PlySs2 is more tractable and stable than previously developed lysins.PlySs2 preparation is straightforward, yielding large quantities in fewsteps. In addition, a highly soluble lysin is essential, because itallows for ready use at high concentrations. Remarkably, PlySs2 remainssoluble in concentrations exceeding 20 mg/ml (data not shown) which isorders of magnitude more concentrated than needed to produce log-foldkilling of target organisms in a matter of minutes. PlySs2 also behaveswell in a variety of in vitro assays developed to evaluate lysins. Itcan be subjected to high or low temperatures for prolonged periods withlittle affect on its activity even when repeatedly freeze-thawed.PlySs2's stability is highly suitable for production and distribution asa therapeutic.

The lack of DTT having an effect on PlySs2 activity indicates both that[1] the lysin does not rely on disulfide bridges, and [2] it wasproperly folded following recombinant expression and purification. Thelatter point is significant given that LySMP had to be treated withreducing agents prior to use [54]. The reason for this discrepancybetween two homologous lysins is unclear, although it may involve thenumerous variable cysteine residues between the proteins. TheEDTA-induced inhibition of PlySs2 suggests that it may rely upon adivalent cation as a cofactor.

Two lysins have been previously shown to have lytic activity against anumber of species, but neither lysin was tested against more than onespecies in vivo [65, 66] and each lysin retained more activity againstthe species (Enterococcus or B. anthracis) from which it was cloned,whereas PlySs2 has more activity against S. aureus than S. suis.

Consistent with its novel broad lytic activity, PlySs2 represents adivergent class of prophage lytic enzyme structure. CHAP domains areincluded in several previously characterized streptococcal [59, 67] andstaphylococcal [50, 68] phage lysins. On a primary sequence level,however, the CHAP domain of PlySs2 is rather divergent from otherdatabase CHAP domains (all pairwise E-values >10⁻¹⁵). In FIG. 30, theCHAP domain of PlySs2 is aligned with that of the well-characterizedstreptococcal PlyC lysin, demonstrating conserved catalytic residues,but only a modest level of identity overall (28% sequence identity,E-value=10⁻⁸) [59].

CHAP domains are catalytically diverse and can possess eitheralanine-amidase [47] or cross-bridge endopeptidase activity [50],depending on the particular lysin. Further, the molecular nature of thepeptidoglycan cross-bridge in S. suis can vary between strains [69]. TheCHAP domain mediates the catalytic activity of PlySs2, but may not fullyconfer specificity. It has been thought that the phage lysin bindingdomains determine lysin specificity [70, 71]. Lysostaphin contains anSH3-like binding domain that presumably binds the cross-bridges in thebacterial cell wall peptidoglycan [71]. SH3 domains are commonly seen inviral and bacterial cell wall-binding proteins, although the exactmolecular target remains unknown [72]. SH3b (bacterial homologues of SH3domains) have been shown to bind metals and polypeptides [73, 74]. AnSH3b domain of a B. cereus endopeptidase has been shown to bind the freeamine group of the N-terminal alanine in the peptidoglycan stem [75] andthis amine group is possible substrate for the PlySs2 SH3b domain. Thecross-bridge varies greatly across all PlySs2-susceptible specimens, soit is an unlikely target for the PlySs2 SH3 binding domain. Thesecross-bridges can acquire variations leading to lysostaphin resistance,however PlySs2 notably displayed activity against bothlysostaphin-sensitive and lysostaphin-resistant (LyrA) S. aureusstrains.

PlySs2 has activity against two, distinct phylogenetic orders:Bacillales (Staphylococcus, Listeria, et al.) and Lactobacillales(Streptococcus, Enterococcus, et al.). The peptidoglycan stems aresimilar between these two orders, but their cross-bridges vary widely incomposition and length [76, 77]. Phage lysins have not previouslydisplayed activity on different families or genera (and rarely ondifferent species) [50]. Native phage lysins usually show speciesspecificity toward the species the native phage infects [48, 61]. All S.aureus strains tested, including strains resistant to methicillin(MRSA), vancomycin (VISA), mupirocin, daptomycin, and lysostaphin weresusceptible to PlySs2-induced lysis. PlySs2 lysed the pathogenic S. suis7997 with similar efficacy. Lysing only 2 of 6 strains tested, PlySs2activity against Listeria was less determinate. The activity of PlySs2against Staphylococus simulans, Streptococcus equi zooepidemicus, andStaphylococcus equi provides further evidence that the substrate for thebinding domain exists outside of the cross-bridge. The polysaccharidecapsule around S. agalactiae enhances its virulence. Type II S.agalactiae has a thicker capsule than most, and has a correspondinglyhigher level of virulence [28]. S. agalactiae strains with a thinnercapsule are less virulent [29, 78]. PlySs2 has comparable activityagainst those with and without a capsule: S. agalactiae_Type II, S.agalactiae 090R. There are greater than 200 M types for S. pyogenes [4].Remarkably, PlySs2 has activity against all of the M-types we tested itagainst. PlySs2 activity against S. sanguinis indicates its potential totreat dental plaque. Although PlySs2 displays activity against S.gordonii, it displays less activity against the commensals S. oralis,and S. mutans. There is a moderate amount of activity against group GStreptococcus, group E Streptococcus, E. facaelis, S. pneumoniae, S.rattus, and S. sobrinus. Other strains of S. pneumoniae were lesssusceptible. The activity of PlySs2 against an array ofmultidrug-resistant MRSA, heavily capsulated S. pyogenes, and numerousother virulent pathogens make it a critical therapeutic candidate.Remarkably, PlySs2 was able to reduce the CFU's of various strains thatvaried in drug resistance, capsulation, and biofilm creation. PlySs2'sactivity against the mucoid S. pyogenes M4 was stronger than wasobserved for any other S. pyogenes tested. Further, PlySs2 had a greateractivity against S. agalactiae and L. monocytogenes than S. pyogenes(from which it was able to protect septicemic mice). Given the efficacyof PlySs2 against S. pyogenes, PlySs2 can serve as a therapeutic agentagainst any of these other pathogens. The MIC results confirmed the CFUfindings.

In murine studies, singly infected animals demonstrate that PlySs2 canindependently protect against multiple species of bacterial infection.In the mixed infection, specific lysin controls (PlyC and ClyS) showPlySs2 is protecting the mice from both organisms in the mixed or duelinfection. Using either PlyC or ClyS to treat only one of the infectiouspathogens in the mixed model still resulted in the death of the animals.Further, the animals die in the same time frame as the singly infectednon-treated controls. S. aureus and S. pyogenes cause diseases withsimilar pathologies and sites of infection in man. Healthcare providersare sometimes not sure at first which organism is causing disease whichcould be a mixed infection in severe trauma cases. Severe invasive S.pyogenes infections are not easily treated with antibiotics [82]. Insome cases, they require surgical procedures [82]. The M1 serotype usedin our septicemic model is one of the leading clinical isolates foundcausing streptococcal pharyngitis and invasive disease worldwide [83,84]. S. pyogenes is able to traverse epithelial surfaces producinginvasive bacteremia [83]. This and other severe internal infectionsresult in death in less than a week in 19% of the cases [85]. In manycases, one does not know whether S. aureus or S. pyogenes is thecausative pathogen behind SSTIs or impetigo.

The inability of pathogenic targets (MRSA and S. pyogenes) to establishresistance to PlySs2 is consistent with findings for other lysinsincluding PlyG [61]. To date, a molecule that can break down lysinsexogenously has not been identified. It is unlikely that a pathogenwould be able to readily alter the target site given the nature ofpeptidoglycan. The extremely low probability of resistance makes PlySs2a compelling therapeutic. Mupirocin and polysporin are typically givento treat S. aureus, but it can develop resistance to each. They are theonly anti-infectives given to reduce colonizing pathogenic bacteria onmucous membranes [86]. PlySs2 can be used to prophylactically clearhuman mucous membrane reservoirs of pathogenic bacteria resistant toantibiotics. Penicillin can be used to treat S. pyogenes, which remainsacutely sensitive; but if the impetigo is caused by MRSA, penicillin maybe ineffective.

Recent studies have indicated that secondary infections caused by S.pneumococcus, S. pyogenes, and MRSA account for >90% of deaths frominfluenza pandemics [92, 93]. The same pathogens caused complications innearly 30% of the 2010 H1N1 pandemic cases [94]. Prophylactic usagecould decrease the rate of these fatalities. PlySs2 may treat primaryinfections and prophylactically decrease the likelihood of secondaryinfections.

Before identification of the pathogen, standard of care is to treat themost likely candidates given the nature and environment of the infection[95, 96]. Many of these pathogens, especially MRSA, readily developresistance to traditional and novel antibiotics, especiallybeta-lactams. MRSA is also resistant to newer agents, glycopeptides,oxazolidinones [97, 98]. PlySs2 has specificity to Gram-positives, butcould broadly treat S. pyogenes, MRSA, and other prominent pathogens.

As a treatment, PlySs2 would target the pathogens, without harmingGram-negatives. This novel capability lies in the divergent CHAP domain,and unique SH3 binding domain. Antibiotics kill many species in additionto the target pathogen. Previously, lysins could only be used againstone pathogenic species. PlySs2 occupies a vital space in the spectrumbetween the rigid lysin specificity, and unselective antibioticactivity. Ideally, a therapeutic has activity against all the majorpathogens without affecting the commensals; PlySs2 is the first toindicate that a lysin could serve that function. PlySs2 is a lysin withbroad lytic activity against MRSA, VISA, S. suis, Listeria, S. simulans,S. equi zoo, S. equi, S. agalactiae, S. pyogenes, S. sanguinis, S.gordonii, group G Streptococcus, group E Streptococcus, E. faecalis, andS. pneumoniae. PlySs2 is easy to produce, tractable, and very stable.PlySs2 protects septicemic mice from a mixed infection of MRSA and S.pyogenes and neither of these pathogens were able to establishresistance to PlySs2.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

1.-42. (canceled)
 43. A method for killing Staphylococcus andStreptococcus bacteria or for reducing a population of Staphylococcusand Streptococcus bacteria comprising the step of contacting thebacteria with a chimeric protein comprising the binding domain of theisolated lysin polypeptide comprising the amino acid sequence of SEQ IDNO:3 or variants thereof having at least 80% identity to the polypeptideof SEQ ID NO:3 and effective to kill Staphylococcus and Streptococcusbacteria, said binding domain operably linked or covalently attached toa heterologous protein or polypeptide, wherein the chimeric protein isbiologically active to bind Staphylococcus and Streptococcus bacteriaand is contacted in an amount effective to kill the Staphylococcus andStreptococcus bacteria.
 44. The method of claim 43 wherein the bindingdomain comprises SEQ ID NO:5 or variants thereof having at least 80%identity to the polypeptide of SEQ ID NO:5 and biologically active tobind Staphylococcus and Streptococcus bacteria.
 45. The method of claim43 wherein the chimeric protein is a chimeric lytic enzyme comprisingthe binding domain of the isolated lysin polypeptide comprising theamino acid sequence of SEQ ID NO:3 or variants thereof having at least80% identity to the polypeptide of SEQ ID NO:3 and effective to killStaphylococcus and Streptococcus bacteria, wherein said binding domainis operably linked to a catalytic domain of another lysin.
 46. Themethod of claim 45 wherein the binding domain of the chimeric lyticenzyme is operably linked to a catalytic domain of aStaphylococcus-specific lysin or wherein the binding domain is operablylinked to a catalytic domain of a Streptococcus-specific lysin.
 47. Themethod of claim 43 wherein the bacteria is an antibiotic resistantbacteria.
 48. The method of claim 43 wherein the bacteria ismethicillin-resistant Staphylococcus aureus (MRSA), vancomycinintermediate-sensitivity Staphylococcus aureus (VISA), or vancomycinresistant Staphylococcus aureus (VRSA).
 49. A method for treating anantibiotic-resistant Staphylococcus aureus infection in a human,comprising the step of administering to the human a chimeric proteincomprising the binding domain of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:3 andeffective to kill Staphylococcus and Streptococcus bacteria, saidbinding domain operably linked or covalently attached to a heterologousprotein or polypeptide, wherein the chimeric protein is biologicallyactive to bind Staphylococcus and Streptococcus bacteria, in an amounteffective to treat the infection.
 50. The method of claim 49 wherein thebinding domain comprises SEQ ID NO:5 or variants thereof having at least80% identity to the polypeptide of SEQ ID NO:5 and biologically activeto bind Staphylococcus and Streptococcus bacteria.
 51. The method ofclaim 49 wherein the chimeric protein is a chimeric lytic enzymecomprising the binding domain of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:3 andeffective to kill Staphylococcus and Streptococcus bacteria, whereinsaid binding domain is operably linked to a catalytic domain of anotherlysin.
 52. The method of claim 51 wherein the binding domain of thechimeric lytic enzyme is operably linked to a catalytic domain of aStaphylococcus-specific lysin.
 53. The method of claim 49 wherein thebacteria is methicillin-resistant Staphylococcus aureus (MRSA),vancomycin intermediate-sensitivity Staphylococcus aureus (VISA), orvancomycin resistant Staphylococcus aureus (VRSA).
 54. A method fortreating gram-positive bacterial infection or for reducing orcontrolling gram-positive bacterial infection or contamination caused byStaphylococcus strains, Streptococcus strains, Listeria monocytogenes orEnterococcus faecalis bacteria in a human, comprising the step ofadministering to the human an effective amount of a chimeric proteincomprising the binding domain of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:3 andeffective to kill Staphylococcus and Streptococcus bacteria, saidbinding domain operably linked or covalently attached to a heterologousprotein or polypeptide, wherein the chimeric protein is biologicallyactive to bind Staphylococcus and Streptococcus bacteria, whereby thenumber of Staphylococcus, Streptococcus, Listeria monocytogenes orEnterococcus faecalis bacteria in the human is reduced and the infectionis controlled.
 55. The method of claim 54 wherein the bacteria isselected from Staphylococcus aureus, Staphylococcus simulans,Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi,Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS),Streptococcus sanguinis, Streptococcus gordonii, Streptococcusdysgalactiae, Streptococcus GES and Streptococcus pneumonia.
 56. Themethod of claim 54 wherein the binding domain comprises SEQ ID NO:5 orvariants thereof having at least 80% identity to the polypeptide of SEQID NO:5 and biologically active to bind Staphylococcus and Streptococcusbacteria.
 57. The method of claim 54 wherein the chimeric protein is achimeric lytic enzyme comprising the binding domain of the isolatedlysin polypeptide comprising the amino acid sequence of SEQ ID NO:3 orvariants thereof having at least 80% identity to the polypeptide of SEQID NO:3 and effective to kill Staphylococcus and Streptococcus bacteria,wherein said binding domain is operably linked to a catalytic domain ofanother lysin.
 58. The method of claim 54 wherein the chimeric proteinis formulated as a topical or dermatological composition foradministration to the skin or external surface of a human.
 59. Themethod of claim 54, further comprising administering one or moreantibiotic.
 60. A method for treating a human subject exposed to or atrisk for exposure to a pathogenic Staphylococcus strain, Streptococcusstrain, Listeria monocytogenes or Enterococcus faecalis bacteria,comprising the step of administering to the human subject a chimericprotein comprising the binding domain of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:3 andeffective to kill Staphylococcus and Streptococcus bacteria, saidbinding domain operably linked or covalently attached to a heterologousprotein or polypeptide, wherein the chimeric protein is biologicallyactive to bind Staphylococcus and Streptococcus bacteria, in an amounteffective to kill the Staphylococcus, Streptococcus, Listeriamonocytogenes or Enterococcus faecalis bacteria.
 61. The method of claim60 wherein the subject is exposed to or at risk of Staphylococcusaureus, Group B Streptococcus bacteria (GBS), Streptococcus pyogenes(GAS) or Streptococcus pneumonia.
 62. The method of claim 60 wherein thebinding domain comprises SEQ ID NO:5 or variants thereof having at least80% identity to the polypeptide of SEQ ID NO:5 and biologically activeto bind Staphylococcus and Streptococcus bacteria.
 63. The method ofclaim 60 wherein the chimeric protein is a chimeric lytic enzymecomprising the binding domain of the isolated lysin polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or variants thereofhaving at least 80% identity to the polypeptide of SEQ ID NO:3 andeffective to kill Staphylococcus and Streptococcus bacteria, whereinsaid binding domain is operably linked to a catalytic domain of anotherlysin.
 64. The method of claim 60 wherein the chimeric protein isformulated as a topical or dermatological composition for administrationto the skin or external surface of a human.
 65. The method of claim 60,further comprising administering one or more antibiotic.